Electrochemical sensors

ABSTRACT

Systems and methods are provided for detecting the presence of an analyte in a sample. A solid state electrochemical sensor can include a redox active moiety having an oxidation and/or reduction potential that is sensitive to the presence of an analyte immobilized over a surface of a working electrode. A redox active moiety having an oxidation and/or reduction potential that is insensitive to the presence of the analyte can be used for reference. Voltammetric measurements made using such systems can accurately determine the presence and/or concentration of the analyte in the sample. The solid state electrochemical sensor can be robust and not require calibration or re-calibration.

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.13/329,135 filed Dec. 16, 2011 which claims the benefit the benefitunder 35 USC 119(e) of U.S. Provisional Patent Application Ser. No.61/424,040, filed Dec. 16, 2010, and U.S. Provisional Patent ApplicationSer. No. 61/550,355, filed Oct. 21, 2011, which are entirelyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The measurement of analyte concentration, in particular, hydrogen ionconcentration or pH is important in a number of research, industrial,and manufacturing processes. For instance, the measurement of pH isroutinely practiced in food and beverage, biofuel, biophamaceuticals, aswell as in the treatment of water and waste.

Many conventional pH sensors use a potentiometric approach whichinvolves the use of glass electrode to measure pH. The potentiometricapproach suffers from several drawbacks. One limitation ofpotentiometric sensors is the need for constant calibration.Potentiometric pH electrodes, like batteries, tend to run down with timeand use. As the potentiometric electrode ages, its glass membrane tendsto change in resistance, which in turn will alter the electrodepotential. For this reason, the glass electrodes require calibration ona regular basis. The need for constant recalibration to provide anaccurate pH output significantly impedes industrial applicationsespecially where constant in-line pH measurements are required.Recalibration is particularly cumbersome in a biotech environment wherepH measurement is conducted in medium containing biological materials.Another significant drawback of conventional pH sensors is that theglass electrodes have internal solutions, which in some cases can leakout into the solution being measured. The glass electrodes can alsobecome fouled by species in the measuring solution, e.g., proteins,causing the glass electrode to foul. ISFET devices have been developedwhich use a field effect transistor structure on a silicon surface tomeasure pH (Bergveld Em et al., IEEE Sensor Conference, Toronto, Oct.2003, 1-26). These devices also have limitations. Thus, there remains aconsiderable need for reliable and consistent analyte sensors, and inparticular, pH sensors.

SUMMARY OF THE INVENTION

An aspect of the invention provides a sensor for detecting the presenceof an analyte comprising: a semiconductor electrode having a surfacehaving immobilized thereon a redox-active moiety, wherein theredox-active moiety exhibits an oxidation potential and/or a reductionpotential that is sensitive to the presence of the analyte. In someembodiments the analyte is hydrogen ion and the redox-active moiety issensitive to hydrogen ion concentration. In some embodiments the sensorcomprises a plurality of redox-active moieties, wherein at least one ofthe redox-active moieties is sensitive to the presence of an analyte,and at least one other redox-active moiety is substantially insensitiveto the presence of the analyte.

In some embodiments the analyte is hydrogen ion, and the moiety that issubstantially insensitive to the presence of hydrogen ion has asubstituent selected from the group consisting of ferrocene,polyvinylferrocene, viologen, polyviologen, and polythiophene. In someembodiments the moiety that is substantially insensitive to the presenceof hydrogen ion is ferrocene or a derivative of ferrocene.

In some embodiments the analyte is hydrogen, and the redox-active moietythat is sensitive to the presence of the hydrogen ion comprises asubstituent selected from the group consisting of anthracenes, quinones,anthroquinones, phenanthroquinones, phenylene diamines, catechols,phenothiazinium dyes, monoquaternized N-alkyl-4,4′-bipyridinium, RuOx,and Ni(OH)2. In some embodiments the redox-active moiety that issensitive to the presence of hydrogen ion comprises a substituentcomprising anthracene. In some embodiments the redox-active moiety thatis sensitive to the presence of hydrogen ion comprises a substituentcomprising an anthraquinone or a phenanthraquinone.

In some embodiments the redox-active moiety that is sensitive to thepresence of an analyte is sensitive to the concentration of the analyte.In some embodiments the oxidation potential and/or reduction potentialof the redox-active moiety is sensitive to the concentration of theanalyte in a range from 10⁻³ M to 10⁻¹⁰ M. In some embodiments oxidationpotential and/or reduction potential of the redox-active moiety issensitive to the concentration of the analyte in a range from 10⁻¹ M to10⁻¹⁴ M. In some embodiments the sensor detects hydrogen ionconcentration from pH 3 to pH 10. In some embodiments the sensor detectshydrogen ion concentration from pH 1 to pH 14.

In some embodiments the sensor measures hydrogen ion concentrationwithin an accuracy of plus or minus 0.1 pH unit. In some embodiments thesensor measures hydrogen ion concentration within an accuracy of plus orminus 0.01 pH units.

In some embodiments the redox-active moiety is covalently bound to thesurface of the electrode. In some embodiments the redox-active moiety isbound to a polymer that is immobilized onto the surface of theelectrode.

In some embodiments the semiconductor electrode is doped. In someembodiments the semiconductor electrode is P-doped. In some embodimentsthe semiconductor electrode comprises a silicon electrode doped withboron. In some embodiments the semiconductor electrode is N-doped. Insome embodiments the semiconductor electrode comprises a siliconelectrode is doped with phosphorous.

In some embodiments the semiconductor electrode comprises a monolithicpiece of silicon.

In some embodiments the semiconductor electrode comprises a compositeelectrode, the composite electrode comprising semiconductor particles ina matrix. In some embodiments the semiconductor electrode comprises acomposite electrode bound to a conductive substrate. In some embodimentsthe semiconductor particles are present in an amount such that aconductive path is created across the composite electrode.

In some embodiments the electrode comprises silicon. In some embodimentsthe electrode comprises unpolished silicon. In some embodiments theelectrode comprises polished silicon.

In some embodiments the semiconductor electrode has a resistivity withinthe range of 0.01 to 1000 Ω-cm. In some embodiments the semiconductorelectrode has a resistivity within the range of 1 to 100 Ω-cm.

In some embodiments the sensor is capable of measuring analyteconcentration without calibration with an external standard. In someembodiments the sensor remains sensitive to the analyte withoutcalibration after a first use by an end user. In some embodiments theanalyte is hydrogen ion and the sensor remains sensitive to hydrogen ionafter exposure to a cell culture medium for at least 6 days. In someembodiments the sensor is capable of measuring pH with an accuracy ofplus or minus 0.2 pH units after exposure to the cell culture medium forat least 6 days. In some embodiments the analyte is hydrogen ion and thesensor is capable of measuring pH with an accuracy of plus or minus 0.2pH units after autoclave treatment at 121° C. for 40 minutes. In someembodiments the sensor is capable of measuring pH with an accuracy ofplus or minus 0.2 units after autoclave treatment at 121° C. for 400minutes.

In some embodiments the semiconductor substrate has a plurality of zoneswherein at least a first zone is sensitive to an analyte, and a secondzone that is insensitive to an analyte.

Another aspect of the invention provides an analyte-sensing systemcomprising: a working electrode having a semiconductor surface that hasimmobilized thereon a redox-active moiety, wherein the redox-activemoiety has an oxidation potential and/or reduction potential that issensitive to the presence of the analyte; a counter electrode andoptionally a reference electrode; a source for supplying a plurality ofpotentials to the working electrode; and a device for measuring currentthrough the working electrode at the plurality of potentials.

In some embodiments the invention further comprises a second workingelectrode comprising a second semiconductor substrate comprising asecond redox-active moiety having an oxidation potential and/orreduction potential that is insensitive to the presence of the analyte.In some embodiments the source for supplying a plurality of potentialsis a potentiostat capable of applying square waves for square wavevoltammetry.

In some embodiments the invention further comprises a computation systemthat communicates with the device for measuring current, and thatcalculates a reduction or oxidation potential from the measured currentat a plurality of potentials.

In some embodiments the system is used as an in-line sensor in aprocess.

In some embodiments the currents measured at a plurality of potentialsare used to determine analyte concentration, and the determined analyteconcentration is used to control a process parameter.

In some embodiments the sensor measures hydrogen ion concentrationwithin an accuracy of plus or minus 0.1 pH unit. In some embodiments thesensor measures hydrogen ion concentration within an accuracy of plus orminus 0.01 pH units.

In some embodiments the sensor is capable of measuring analyteconcentration without calibration with an external standard. In someembodiments the sensor remains sensitive to the analyte withoutcalibration after a first use by an end user. In some embodiments theanalyte is hydrogen ion and the sensor remains sensitive to hydrogen ionafter exposure to a cell culture medium for at least 6 days. In someembodiments the sensor is capable of measuring pH with an accuracy ofplus or minus 0.2 units after exposure to the cell culture medium for atleast 6 days. In some embodiments the analyte is hydrogen ion and thesensor is capable of measuring pH with an accuracy of plus or minus 0.2units after autoclave treatment at 121° C. for 40 minutes. In someembodiments the sensor is capable of measuring pH with an accuracy ofplus or minus 0.2 units after autoclave treatment at 121° C. for 400minutes.

Another aspect of the invention provides a semiconductor substratehaving a surface, wherein the surface comprises a redox-active moietyimmobilized thereon, the redox-active moiety exhibiting an oxidationpotential and/or a reduction potential that is sensitive to the presenceof an analyte.

In some embodiments immobilized thereon is also a second redox-activemoiety having an oxidation potential and/or a reduction potential thatis substantially insensitive to the presence of the analyte.

In some embodiments the analyte is an ion. In some embodiments theanalyte is hydrogen ion. In other embodiments, the analyte is apolarizable molecule. In other embodiments, the analyte is an ionizablemolecule, such as upon the application of an external source of energy.

In some embodiments the semiconductor comprises an inorganicsemiconductor. In some embodiments the semiconductor comprises anorganic semiconductor. In some embodiments the inorganic semiconductorcomprises silicon or gallium arsenide. In some embodiments the organicsemiconductor comprises polyacetylene, polythiophene, or polypyrrole.

In some embodiments the semiconductor comprises silicon. In someembodiments the semiconductor comprises unpolished silicon. In someembodiments the semiconductor comprises polished silicon.

In some embodiments the redox-active moiety is directly bound to thesurface. In some embodiments the redox-active moiety is covalently boundto the surface. In some embodiments the redox-active moiety iscovalently bound to the surface through a linker. In some embodimentsthe redox-active moiety is covalently bound to a polymer that isimmobilized onto the surface of the semiconductor substrate. In someembodiments the redox-active moiety is covalently bound to a polymerthat is covalently bound to the surface of the semiconductor substrate

In some embodiments the semiconductor is doped. In some embodiments thesemiconductor is N-doped. In some embodiments the semiconductor isP-doped.

In some embodiments substrate comprises crystalline silicon wherein thesurface displaying predominantly one crystalline plane.

In some embodiments the substrate has a plurality of zones wherein eachzone comprises a redox-active moiety immobilized thereon, theredox-active moiety exhibiting an oxidation potential and/or a reductionpotential that is sensitive to the presence of an analyte. In someembodiments a first zone comprises a redox moiety sensitive to a firstanalyte, and a second zone comprises a redox moiety sensitive to asecond analyte. In some embodiments the invention further comprises athird redox-active moiety exhibiting an oxidation potential and/or areduction potential that is sensitive to the presence of a secondanalyte. In some embodiments the second analyte is ammonia, oxygen orcarbon dioxide.

Another aspect of the invention provides a method for forming ananalyte-sensitive semiconductor electrode, the electrode having asurface, the method comprising immobilizing a redox-active moiety thatis sensitive to the presence of an analyte onto the surface. In someembodiments immobilizing the redox-active moiety covalently binds theredox-active moiety to the surface. In some embodiments the redox-activemoiety is covalently bound to the surface through a linker. In someembodiments immobilizing the redox-active moiety compriseshydrosilation, a free radical reaction, carbodiimide coupling, aDiels-Alder reaction, a Michael addition, or click chemistry.

In some embodiments the redox-active moiety is covalently bound to apolymer that is immobilized onto the surface.

In some embodiments the step of immobilizing comprises polymerizationincluding a monomer or oligomer comprising a redox-active moiety. Insome embodiments the polymerization of a monomer or oligomer comprisinga redox-active moiety includes a reaction with a functional groupcovalently bound to the surface, whereby the polymer formed bypolymerization is covalently bound to the surface. In some embodimentsthe monomer or oligomer is electropolymerized onto the surface.

In some embodiments the step of immobilizing comprises coating orcasting the polymer onto the surface.

In some embodiments the semiconductor surface comprises a compositeelectrode that comprises semiconductor particles within a matrix.

Another aspect of the invention provides a method for forming asemiconductor surface derivatized with one or more redox-active moietiescomprising: contacting an H-terminated semiconductor surface with theone or more redox-active moieties wherein at least one redox activemoiety is sensitive to the presence of an analyte, and wherein eachredox-active moiety comprises a functional group that will react withthe H-terminated semiconductor surface to form a covalently bond,thereby forming a derivatized semiconductor surface.

In some embodiments the semiconductor surface comprises silicon.

In some embodiments at least two redox active moieties are used, and oneof the redox active moieties is insensitive to the presence of theanalyte.

In some embodiments the H-terminated semiconductor surface is formed bytreatment with hydrofluoric acid.

In some embodiments the functional group is a vinyl group. In someembodiments the functional group is an aldehyde group. In someembodiments the functional group is a diazonium group.

Another aspect of the invention provides a method of determining theconcentration of an analyte, comprising placing an electrode in contactwith the analyte, the electrode comprising a semiconductor substratewith a semiconductor surface having immobilized thereon ananalyte-sensitive redox-active moiety, the analyte-sensitiveredox-active moiety exhibiting an oxidation potential and/or reductionpotential that is sensitive to the concentration of the analyte;applying a plurality of potentials to the electrode; and measuring thecurrent through the electrode at the plurality of potentials todetermine a reduction and/or oxidation potential of theanalyte-sensitive redox-active moiety, thereby determining theconcentration of the analyte.

In some embodiments the measuring of the current at the plurality ofpotentials provides a peak current, and whereby the peak current is usedto determine reduction and/or oxidation potential of theanalyte-sensitive redox-active moiety.

In some embodiments the analyte is hydrogen ion.

In some embodiments the invention further comprises ananalyte-insensitive redox-active moiety bound to a an electrodecomprising a semiconductor substrate, such redox-active moiety having areduction and/or oxidation potential that is substantially insensitiveto the analyte, further comprising determining the oxidation and/orreduction potential of the analyte-insensitive redox-active moiety, anddetermining the concentration of the analyte from the difference in theoxidation and/or reduction potentials of the analyte-sensitive andanalyte-insensitive moieties.

In some embodiments the analyte is provided in a solution.

Another aspect of the invention provides a sensor for detecting thepresence of an analyte. The sensor comprises a semiconductor electrodehaving a surface having immobilized thereon a layer of a redox-activemoiety, wherein the redox-active moiety exhibits an oxidation potentialand/or a reduction potential that is sensitive or insensitive to thepresence of the analyte, and a layer of composite material on, over oradjacent to the layer of the redox-active moiety. In some cases, thelayer of the composite material covers the layer of the redox-activemoiety.

In some embodiments, the composite material comprises Nafion. In somecases, the composite material comprises a porous material, such as aporous polymeric material (e.g., plastic), impregnated with Nafion.

In some situations, a working electrode comprises a layer of a polymericmaterial for shielding light-sensitive moieties on or over the workingelectrode from light. In some cases, the layer of the polymeric materialcomprises polyethersulphone (PES).

In another aspect of the invention, an electrochemical sensor comprisesa solid state (e.g., a semiconductor, such as silicon, or carbon)electrode that is equipped with (or operatively coupled to) a lightemitting device (also “light source” herein).

In some embodiments, a sensor comprises a light-emitting device, such asa light-emitting diode, and a layer of a semiconducting or anon-semiconducting material over the light emitting device. The layer ofthe semiconducting or non-semiconducting material may have a redoxsensitive moiety thereon.

In some embodiments, the light-emitting diode is an organiclight-emitting diode. In some embodiments, the semiconducting materialis silicon. In some embodiments, the non-semiconducting material iscarbon, such as activated carbon.

In another embodiment, a sensor for detecting the presence or absence ofan analyte comprises an electrode having a surface having immobilizedthereon a redox-active moiety, wherein the redox-active moiety exhibitsan oxidation potential and/or a reduction potential that is sensitive orinsensitive to the presence of the analyte. A light-emitting device isadjacent to the electrode. The light-emitting device is configured togenerate light.

In some embodiments, a solid state sensor for detecting the presence orabsence of an analyte, comprises a solid state electrode configured todetect the presence or absence of the analyte, and a light emittingdevice adjacent to the solid state electrode.

In some embodiments, a sensor for detecting the presence or absence ofan analyte, comprising a working electrode having a redox active moietyformed adjacent a light emitting device, is provided.

In another aspect of the invention, a sensor for detecting the presenceor absence of an analyte comprises a semiconductor electrode having asurface having immobilized thereon a layer of a redox-active moiety,wherein the redox-active moiety exhibits an oxidation potential and/or areduction potential that is sensitive or insensitive to the presence ofthe analyte. A light blocking layer is adjacent to the layer of theredox-active moiety.

In some embodiments, a solid state sensor for detecting the presence orabsence of an analyte, comprises a solid state electrode and a lightblocking layer adjacent to the solid state electrode. The light blockinglayer may be formed of a polymeric material. In some situations, thelight blocking layer transmits less than 10%, 5%, or 1% of lightincident on the light blocking layer.

Another aspect of the invention is a method comprising: measuring a pHvalue of a step in a water or waste treatment process with avoltammetric pH sensor, wherein the pH sensor comprises a redox-activemoiety that is sensitive to hydrogen ion concentration, and aredox-active moiety that is substantially insensitive to hydrogen ion;and using the pH value to monitor or control the treatment process.

Another aspect of the invention provides a method comprising measuring apH value of a reaction mixture in a biopharmaceutical process with avoltammetric pH sensor, wherein the pH sensor comprises a redox-activemoiety that is sensitive to hydrogen ion concentration, and aredox-active moiety that is substantially insensitive to hydrogen ionconcentration to obtain a pH value. The pH value is used to monitor thebiopharmaceutical process.

In some embodiments the pH value is measured on a sample obtained fromthe reaction mixture.

Another aspect of the invention provides a reactor for carrying out abiopharmaceutical process wherein the reactor comprises a pH sensorhaving a redox-active moiety that is sensitive to hydrogen ionconcentration, and a redox-active moiety that is substantiallyinsensitive to hydrogen ion concentration.

In some embodiments the pH sensor is a voltammetric pH sensor.

In an embodiment, the pH sensor is a disposable pH sensor. In anotherembodiment, the pH sensor is a single-use pH sensor. In anotherembodiment, the pH sensor is a disposable and single-use pH sensor.

In some embodiments the reactor is a disposable bioreactor. In someembodiments the reactor is a bioprocess flexible container.

Another aspect of the invention provides a method for carrying out anindustrial process comprising: measuring a pH value of a step of anindustrial process with a voltammetric pH sensor having a redox-activemoiety that is sensitive to hydrogen ion concentration, and aredox-active moiety that is substantially insensitive to hydrogen ionconcentration; and using the pH value to carry out the industrialprocess.

Another aspect of the invention provides a sensor for measuring ionconcentration in a bodily fluid within a body. The sensor comprises anelectrode configured to be in contact with a bodily fluid, the electrodecomprising a semiconductor surface that has immobilized thereon aredox-active moiety. The redox-active moiety has an oxidation potentialand/or reduction potential that is sensitive to concentration of theion.

Another aspect of the invention provides a method for measuringconcentration in a bodily fluid within a body, comprising placing such asensor in contact with the bodily fluid and operating the sensor toyield a value of the concentration of the ion present in the bodilyfluid.

Another aspect of the invention provides a bioreactor comprising areservoir for containing a reaction mixture and a pH probe wherein thepH probe comprises an electrode having a semiconductor surface, thesemiconductor surface having immobilized thereon a redox active moietyhaving a reduction and/or oxidation potential that is sensitive to thepresence of hydrogen ion.

In some embodiments the invention further comprises a semiconductorsurface having immobilized thereon a redox active moiety having areduction and/or oxidation potential that is insensitive to the presenceof hydrogen ion.

In some embodiments the semiconductor surface on which the redox activemoiety having a reduction and/or oxidation potential that is sensitiveto the presence of hydrogen ion is immobilized is the same semiconductorsurface on which the redox active moiety having a reduction and/oroxidation potential that is insensitive to the presence of hydrogen ionis immobilized on. In some embodiments the probe further comprises acounter electrode.

Another aspect of the invention provides a sensor system, comprising aredox-active moiety-containing analyte sensor for insertion into acontainer for use with a glass probe analyte sensor. In an embodiment,the redox-active moiety-containing analyte sensor comprises one or moreredox-active moieties. In another embodiment, the redox-activemoiety-containing analyte sensor comprises a redox-active moiety that issensitive to the presence of an analyte and another redox-active moietythat is insensitive to the presence of the analyte. In anotherembodiment, the redox-active moiety-containing analyte sensor isdisposed in a probe body having a form factor configured for insertioninto a container for use with a glass probe analyte sensor.

Another aspect of the invention provides a method for detecting thepresence or absence of an analyte, comprising using a sensor system, asdescribed above, to detect the presence or absence of the analyte.

Another aspect of the invention provides a method for forming an analytesensor, comprising inserting a sensor system as described herein into acontainer configured for use with a glass probe analyte sensor. In anembodiment, the method further comprises removing a glass probe analytesensor from the container prior to inserting the sensor system into thecontainer.

Another aspect of the invention provides a method for forming an analytesensor, comprising inserting a sensor system as described herein into acontainer configured for use with a reactor, in-line flow system, orsample preparation, or analysis.

Another aspect of the invention provides a sensor for detecting thepresence or absence of an analyte, comprising a semiconductor electrodehaving a surface having immobilized thereon a redox-active moiety,wherein the redox-active moiety exhibits an oxidation potential and/or areduction potential that is sensitive to the presence of the analyte.The sensor includes a form factor for insertion into a container of aglass probe analyte sensor.

In an embodiment, the analyte is hydrogen ion and the redox-activemoiety is sensitive to hydrogen ion concentration. In anotherembodiment, the sensor comprises a plurality of redox-active moieties,wherein at least one of the redox-active moieties is sensitive to thepresence of an analyte, and at least one other redox-active moiety issubstantially insensitive to the presence of the analyte. In anotherembodiment, the analyte is hydrogen ion, and the moiety that issubstantially insensitive to the presence of hydrogen ion has asubstituent selected from the group consisting of ferrocene,polyvinylferrocene, viologen, polyviologen, and polythiophene. Inanother embodiment, the moiety that is substantially insensitive to thepresence of hydrogen ion is ferrocene or a derivative of ferrocene. Inanother embodiment, the analyte is hydrogen, and the redox-active moietythat is sensitive to the presence of the hydrogen ion comprises asubstituent selected from the group consisting of anthracenes, quinones,anthroquinones, phenanthroquinones, phenylene diamines, catechols,phenothiazinium dyes, monoquaternized N-alkyl-4,4′-bipyridinium, RuOx,and Ni(OH)₂. In another embodiment, the redox-active moiety that issensitive to the presence of hydrogen ion comprises a substituentcomprising anthracene. In another embodiment, the redox-active moietythat is sensitive to the presence of hydrogen ion comprises asubstituent comprising an anthraquinone or a phenanthraquinone. Inanother embodiment, the redox-active moiety that is sensitive to thepresence of an analyte is sensitive to the concentration of the analyte.In another embodiment, the oxidation potential and/or reductionpotential of the redox-active moiety is sensitive to the concentrationof the analyte in a range from 10⁻³ M to 10⁻¹⁰ M. In another embodiment,the oxidation potential and/or reduction potential of the redox-activemoiety is sensitive to the concentration of the analyte in a range from10⁻¹ M to 10⁻¹⁴ M.

In an embodiment, the sensor detects hydrogen ion concentration from pH1 to pH 14. In another embodiment, the sensor detects hydrogen ionconcentration from pH 3 to pH 10. In another embodiment, the sensormeasures hydrogen ion concentration within an accuracy of plus or minus0.1 pH units. In another embodiment, the sensor measures hydrogen ionconcentration within an accuracy of plus or minus 0.01 pH units.

In an embodiment, the redox-active moiety is covalently bound to thesurface of the electrode, such as through an oxygen-to-surface,carbon-to-surface or sulfur-to-surface bond. In another embodiment, theredox-active moiety is bound to a polymer that is immobilized onto thesurface of the electrode.

In an embodiment, the semiconductor electrode is doped. In anotherembodiment, the semiconductor electrode is p-doped. In anotherembodiment, the semiconductor electrode is doped with boron. In anotherembodiment, the semiconductor electrode is n-doped. In anotherembodiment, the semiconductor electrode is doped with phosphorous. Inanother embodiment, the semiconductor electrode comprises a monolithicpiece of silicon. In another embodiment, the semiconductor electrodecomprises a composite electrode, the composite electrode comprisingsemiconductor particles in a matrix. In another embodiment, thesemiconductor electrode comprises a composite electrode bound to aconductive substrate. In another embodiment, the semiconductor particlesare present in an amount such that a conductive path is created acrossthe composite electrode. In another embodiment, the electrode comprisessilicon. In another embodiment, the electrode comprises unpolishedsilicon. In another embodiment, the electrode comprises polishedsilicon. In another embodiment, the semiconductor electrode has aresistivity within the range of about 0.01 to 1000 Ω-cm. In anotherembodiment, the semiconductor electrode has a resistivity within therange of 1 to 100 Ω-cm.

In an embodiment, the sensor is capable of measuring analyteconcentration without calibration with an external standard. In anotherembodiment, the sensor remains sensitive to the analyte withoutcalibration after a first use by an end user. In another embodiment, theanalyte is hydrogen ion and the sensor remains sensitive to hydrogen ionafter exposure to a cell culture medium for at least 6 days. In anotherembodiment, the sensor is capable of measuring pH with an accuracy ofplus or minus 0.2 pH units after exposure to the cell culture medium forat least 6 days. In another embodiment, the analyte is hydrogen ion andthe sensor is capable of measuring pH with an accuracy of plus or minus0.2 pH units after autoclave treatment at 121° C. for 40 minutes. Inanother embodiment, the sensor is capable of measuring pH with anaccuracy of plus or minus 0.2 pH units after autoclave treatment at 121°C. for 400 minutes. In another embodiment, the semiconductor substratehas a plurality of zones, wherein at least a first zone is sensitive toan analyte and a second zone is insensitive to an analyte.

In an embodiment, the container is cylindrical in shape. In anotherembodiment, the container has a circular cross-section. In anotherembodiment, the container is formed of one or more metals. In anotherembodiment, the one or more metals include stainless steel.

Another aspect of the invention provides a sensor comprising a solidstate electrode having a surface immobilized thereon a mixed layer ofhydrocarbon molecules and redox-active moieties. In an embodiment, theredox-active moieties are sensitive to an analyte, such as hydrogen. Inanother embodiment, the redox-active moieties are insensitive to theanalyte.

Another aspect of the invention provides a sensor for detecting thepresence or absence of an analyte, comprising a semiconductor electrodehaving a surface having immobilized thereon a layer of a redox-activemoiety, wherein the redox-active moiety exhibits an oxidation potentialand/or a reduction potential that is sensitive or insensitive to thepresence of the analyte. The sensor further comprises a light blockinglayer adjacent to the layer of the redox-active moiety. In anembodiment, the light blocking layer comprises a polymeric material. Inanother embodiment, the polymeric material comprises afluoropolymer-copolymer. In another embodiment, the polymeric materialcomprises Nafion. In another embodiment, the sensor further comprises aprotective layer adjacent to the light blocking layer. In anotherembodiment, the protective layer comprises a polymeric material. Inanother embodiment, the polymeric material comprises polyethersulphone.

In an embodiment, the light blocking layer comprises a compositematerial. In another embodiment, the composite material comprisesNafion. In another embodiment, the composite material comprises a porousplastic. In another embodiment, the composite material is a porousmembrane.

Another aspect of the invention provides a redox-activemoiety-containing analyte sensor for use with a glass probe analytedetection system. The redox-active moiety-containing analyte sensor canhave any of the features and characteristics of sensors described above.

Another aspect of the invention provides a redox-activemoiety-containing analyte sensor for use in a time period of about 1day, or 5 days, or 10 days, or 20 days, or 25 days, or 30 days. Theredox-active moiety-containing analyte sensor can have any of thefeatures and characteristics of sensors described above.

Another aspect of the invention provides a redox-activemoiety-containing analyte sensor having a sensitivity between about 20mV per pH unit and 60 mV per pH unit. The redox-active moiety-containinganalyte sensor can have any of the features and characteristics ofsensors described above.

Another aspect of the invention provides a redox-activemoiety-containing analyte sensor having a shelf life between about 3months and 3 years. The redox-active moiety-containing analyte sensorcan have any of the features and characteristics of sensors describedabove.

Another aspect of the invention provides a sensor for detecting ananalyte having an accuracy to within 0.001 pH units while in use orstorage for at least 2 years. The redox-active moiety-containing analytesensor can have any of the features and characteristics of sensorsdescribed above.

Another aspect of the invention provides a sensor for detecting ananalyte having an accuracy to within 0.001 pH units while in use orstorage for at least 4 years. The redox-active moiety-containing analytesensor can have any of the features and characteristics of sensorsdescribed above.

Another aspect of the invention provides a sensor for detecting ananalyte having an accuracy to within 0.001 pH units while in use orstorage for at least 8 years. The redox-active moiety-containing analytesensor can have any of the features and characteristics of sensorsdescribed above.

Another aspect of the invention provides a sensor for detecting ananalyte having an accuracy to within 0.001 pH units while in use orstorage for at least 10 years. The redox-active moiety-containinganalyte sensor can have any of the features and characteristics ofsensors described above.

Another aspect of the invention provides a sensor for detecting thepresence or absence of an analyte, comprising an electrode having asurface having immobilized thereon a redox-active moiety, wherein theredox-active moiety exhibits an oxidation potential and/or a reductionpotential that is sensitive or insensitive to the presence of theanalyte, and a light-emitting device adjacent to the electrode, thelight-emitting device configured to generate light. In an embodiment,the light-emitting device is configured to generate light that is i)incident on the surface, ii) incident on another surface of theelectrode, the another surface opposite from the surface, and/or iii)directed through the electrode. In another embodiment, the electrode isformed of a solid state material. In another embodiment, the electrodeis formed of a semiconductor material. In another embodiment, thesemiconductor material includes silicon. In another embodiment, theelectrode is formed of a non-semiconductor material. In anotherembodiment, the non-semiconductor material includes carbon. In anotherembodiment, the light-emitting device is a light-emitting diode havingan active region configured to generate light upon the recombination ofelectrons and holes. In another embodiment, the light-emitting diode isan organic light-emitting diode. In another embodiment, during use,light from the light-emitting device is incident on the surface.

Another aspect of the invention provides a sensor for detecting thepresence or absence of an analyte, comprising a working electrode havinga redox active moiety formed adjacent a light emitting device. Thesensor can have any of the features and characteristics of sensorsdescribed above.

Another aspect of the invention provides a solid state sensor fordetecting the presence or absence of an analyte, comprising a solidstate electrode and a light blocking layer adjacent to the solid stateelectrode. In an embodiment, the light blocking layer is formed of apolymeric material. In another embodiment, the light blocking layertransmits less than 10% of light incident on the light blocking layer.In another embodiment, the light blocking layer transmits less than 5%of light incident on the light blocking layer. In another embodiment,the light blocking layer transmits less than 1% of light incident on thelight blocking layer.

Another aspect of the invention provides a solid state sensor fordetecting the presence or absence of an analyte, comprising a solidstate electrode configured to detect the presence or absence of theanalyte, and a light emitting device adjacent to the solid stateelectrode. The sensor can have any of the features and characteristicsof sensors described above.

Another aspect of the invention provides a sensor for detecting thepresence or absence of an analyte, comprising a working electrode havinga redox active moiety formed adjacent a light emitting device. Thesensor can have any of the features and characteristics of sensorsdescribed above.

Another aspect of the invention provides a method for detecting thepresence or absence of an analyte, comprising bringing an analyte sensorin contact with a sample, the analyte sensor having an electrode havingimmobilized thereon a redox-active moiety. The redox-active moietyexhibits an oxidation potential and/or a reduction potential that issensitive to the presence of the analyte. Next, with the aid of theanalyte sensor, the analyte is detected at an accuracy within at leastabout 5% without re-calibration for a period of at least about 1 day. Inan embodiment, the accuracy is within at least about 1%. In anotherembodiment, the accuracy is within at least about 0.1%. In anotherembodiment, the period is at least about 7 days. In another embodiment,the period is at least about 1 month. In another embodiment, the periodis at least about 1 year. In another embodiment, the period is at leastabout 2 years.

Another aspect of the invention provides a method for detecting thepresence or absence of an analyte, comprising using a sensor, asdescribed above, to detect the presence or absence of the analyte. Insome embodiments, the analyte is hydrogen ion.

Another aspect of the invention provides a sensor having a solid stateworking electrode having disposed thereon a redox-active moietyexhibiting an oxidation potential and/or a reduction potential that issensitive to the presence of an analyte. The working electrode has asize and shape for use in glass probe sensor, a reactor, a flow system,or a sample separation system. In an embodiment, the reactor is abioreactor. In another embodiment, the sensor further comprises anadditional working electrode having disposed thereon a redox-activemoiety exhibiting an oxidation potential and/or reduction potential thatis insensitive to the presence of the analyte. In another embodiment,the working electrode is doped p-type and the additional workingelectrode is doped n-type or p-type. In another embodiment, the workingelectrode has a resistivity is greater than or equal to about 1 Ω-cm andthe additional working electrode has a resistivity greater than or equalto about 5 μΩ-cm.

Another aspect of the invention provides an analyte sensor, comprising afirst solid state working electrode and a second solid state workingelectrode. The first solid state working electrode has disposed thereona redox-active moiety exhibiting an oxidation potential and/or areduction potential that is sensitive to the presence of an analyte, thefirst solid state working electrode doped p-type. The second solid stateworking electrode has disposed thereon a redox-active moiety exhibitingan oxidation potential and/or a reduction potential that is insensitiveto the presence of the analyte, the second solid state working electrodedoped n-type or p-type. In an embodiment, the first solid state workingelectrode is disposed adjacent to the second solid state workingelectrode. In another embodiment, the first solid state workingelectrode is electrically isolated from the second solid state workingelectrode. In another embodiment, the first solid state workingelectrode has a resistivity greater than or equal to 1 Ω-cm (also “Ωcm”herein). In another embodiment, the second solid state working electrodehas a resistivity greater than or equal to about 5 μΩ-cm. In anotherembodiment, the first and second solid state working electrodes areformed of a semiconductor. In another embodiment, the semiconductor issilicon. In another embodiment, the second solid state working electrodeis doped n-type and has a resistivity greater than or equal to about 1Ω-cm. In another embodiment, the resistivity of the second solid stateworking electrode is between about 1 Ω-cm and 90 Ω-cm. In anotherembodiment, the solid state working electrodes are disposed on asubstantially flat surface of the analyte sensor.

Another aspect of the invention provides a method for forming an analytesensor, comprising inserting a sensor as described herein into acontainer that is for use with a glass probe analyte sensor. In anembodiment, the method further comprises removing a glass probe analytesensor from the container prior to inserting the sensor into thecontainer.

Another aspect of the invention provides a method for forming an analytesensor, comprising inserting a sensor as described herein into acontainer that is for use with a reactor, in-line flow system, or samplepreparation, or analysis.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the invention may be further explained byreference to the following detailed description and accompanyingdrawings (or figures, also “Fig.” and “FIG.” herein) that sets forthillustrative embodiments.

FIG. 1( a) shows a blow up drawing illustrating an embodiment of theinvention comprising a semiconductor electrode sensor in a housingassembly, in accordance with an embodiment of the invention. FIG. 1( b)shows an exemplary housing assembly comprising the semiconductorelectrode sensor, in accordance with an embodiment of the invention;

FIGS. 2( a) and 2(b) each depict a view of an embodiment of theinvention comprising a unit that electrically connects to thesemiconductor electrode sensor and comprising a source for supplying aplurality of potentials and a current measuring device, in accordancewith an embodiment of the invention;

FIG. 3 depicts an embodiment of the invention comprising a probe formeasuring analytes within a reactor comprising two working siliconelectrodes, in accordance with an embodiment of the invention;

FIG. 4 illustrates a method of preparing of H-terminated silicon surface(Si—H), in accordance with an embodiment of the invention;

FIG. 5 illustrates a silicon surface derivatization with ferrocenemoieties, vinyl-ferrocene (VFc) and ferrocene carboxaldehyde (FcA) bycovalent attachment, in accordance with an embodiment of the invention;

FIG. 6 illustrates a silicon surface derivatized with anthracenemoieties, vinyl anthracene (VA) and anthraldehyde (AnA) by covalentattachment, in accordance with an embodiment of the invention;

FIG. 7 illustrates a silicon surface derivatized with both theanthracene (VA) and ferrocene (VFc) moieties by covalent attachment, inaccordance with an embodiment of the invention;

FIG. 8 depicts a schematic diagram and several picture views of anexemplary electrochemical cell, in accordance with an embodiment of theinvention;

FIG. 9 depicts square wave voltammograms (left) showing the effect of pHon VFc derivatized silicon sample (right) at pH solution of 1.23, 4.61,7.33 and 9.33, in accordance with an embodiment of the invention;

FIG. 10( a) depicts square wave voltammograms (left) showing the effectof pH on VA derivatized silicon sample (right) at pH solutions of 1.23,4.61, 7.33 (not shown; peak maximum between −0.4 and −0.6 V, to the leftof the pH 4.61 peak and to the right of the pH 13.63 peak) and 13.63, inaccordance with an embodiment of the invention. FIG. 10( b) depicts aplot of peak potential against pH using the VA derivatized siliconsample, in accordance with an embodiment of the invention;

FIG. 11 depicts: (a) square wave voltammograms (left) showing the effectof pH on VA+VFc derivatized silicon sample (right) at pH solutions of1.23, 4.65, 5.52 and 9.32 (VA circle, peaks going from right to left);and (b) a plot of peak potential difference against pH using the VA+VFcderivatized silicon sample, in accordance with an embodiment of theinvention;

FIG. 12 depicts square wave voltammograms (left) showing the effect of10 autoclave cycles on FcA+VA derivatized silicon sample (right), inaccordance with an embodiment of the invention. The electrochemicalmeasurements were conducted at pH 6.52 buffer prior to autoclave andafter autoclave;

FIG. 13 depicts square wave voltammograms showing minimal fouling onFcA+AnA derivatized silicon samples, in accordance with an embodiment ofthe invention. The electrochemical measurements were conducted at pH6.52 buffer before and after six days exposure in the cell culture atfour different FcA+AnA derivatized silicon samples (a), (b), (c) and(d);

FIG. 14 depicts a square wave voltammogram obtained at FcA+AnAderivatized silicon sample in cell culture medium after sterilizationand 6 days exposure, in accordance with an embodiment of the invention;

FIG. 15( a) depicts square wave voltammetric responses FcA on Si(100,N-type, 1-5 mΩ cm) in pH 7.33 buffer medium, showing every 50^(th) scanof the 2,500 consecutive runs, in accordance with an embodiment of theinvention. FIG. 15( b) depicts voltammetric responses of VFc on Si(111,N-type, 0.02-0.05Ω cm in pH 7.33 buffer medium, showing every 50^(th)scan of the 2,500 consecutive runs, in accordance with an embodiment ofthe invention;

FIG. 16 depicts the square wave voltammetric response of Ac derivatizedsilicon surface at various temperatures (8, 17, 28 44, 56° C.) in pH7.33 buffer medium, in accordance with an embodiment of the invention;

FIG. 17 is a drawing of an embodiment of a bioreactor of the inventioncomprising a silicon-based voltammetric sensor, in accordance with anembodiment of the invention;

FIG. 18( a) depicts voltammograms taken on an anthracene derivatizedsilicon sensor over the 7 day period in cell culture medium (every250^(th) scan of the 10,000 consecutive runs), in accordance with anembodiment of the invention. FIG. 18( b) depicts a plot of theanthracene peak potential over the 7 day time period, in accordance withan embodiment of the invention;

FIG. 19 has charts showing the peak current of silicon substratesderivatized with (a) vinyl ferrocene and (b) ferrocene carboxaldehyde inpH 1.63 solution for four types of doped silicon, in accordance with anembodiment of the invention;

FIG. 20 depicts square wave voltammograms obtained with a four electrodesystem having highly-doped N-type silicon derivatized with vinylferrocene and at a lightly-doped p-type silicon derivatized withanthracene carboxaldehyde, in accordance with an embodiment of theinvention;

FIG. 21 depicts an exemplary embodiment of a probe of the presentinvention having a four electrode configuration with a ring referenceelectrode (RE) and a ring counter electrode (CE), in accordance with anembodiment of the invention. FIG. 21( a) is a shaded drawing, and FIG.21( b) is a line drawing;

FIG. 22A schematically illustrates an electrochemical sensor havingthree modules; FIG. 22B is an enlarged view of a portion of theelectrochemical sensor of FIG. 22A, in accordance with an embodiment ofthe invention;

FIG. 23 schematically illustrates a printed circuit board, in accordancewith an embodiment of the invention;

FIG. 24 schematically illustrates a sensor formed on a printed circuitboard and mounted on a head assembly, in accordance with an embodimentof the invention;

FIG. 25A schematically illustrates an electrochemical sensor mounted ona wall of a disposable container, in accordance with an embodiment ofthe invention; FIG. 25B illustrates an electrochemical sensor comprisingelectrodes formed on a printed circuit board, in accordance with anembodiment of the invention;

FIG. 26 schematically illustrates an electrochemical sensor mounted on achamber of a flow-through tube, in accordance with an embodiment of theinvention;

FIG. 27A schematically illustrates an electronics unit for permittingintegration of an electrochemical sensor into current probe systems, inaccordance with an embodiment of the invention; FIG. 27B illustrates aprobe attached to the electronics unit of FIG. 27A, in accordance withan embodiment of the invention; FIG. 27C shows a probe attached to anelectronic unit, which is in turn attached to a reader, in accordancewith an embodiment of the invention;

FIG. 28 schematically illustrates an electrochemical probe mounted on abioreactor, in accordance with an embodiment of the invention;

FIG. 29 shows a working electrode having a light-emitting device and anelectrode adjacent to the light-emitting device, in accordance with anembodiment of the invention;

FIG. 30 shows sensor output (y-axis, mV) with time (x-axis) during pHmeasurements for a shielded (top) and unshielded (bottom) sensors;

FIGS. 31A-31F show sensor output (y-axis; current, arbitrary units) atvarious pH's and under light and dark conditions as a function ofvoltage (mV);

FIGS. 32A-32E show exemplary sensors having form factors suited forvarious applications; and

FIG. 33 shows the pH of a fermentation reactor as a function of time asmeasured by a glass electrode sensor and a redox-active moiety-based pHsensor.

DETAILED DESCRIPTION OF THE INVENTION

While preferable embodiments of the invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

The invention relates to compositions, devices, systems, and methods forproducing and using solid state electrodes modified with redox-activeagents as sensors. The subject devices and systems are particularlyuseful for voltammetrically measuring concentrations of an analyte ofinterest. The sensors of the present invention utilize a solid stateelectrode comprising redox-active species on its surface. At least oneredox-active species on the solid sate (e.g., semiconductor) surface hasa redox potential (reduction potential or oxidation potential) that issensitive to the presence and or amount of an analyte of interest.Voltammetry can be performed on the solid state electrode and used tomeasure the redox potential of the analyte-sensitive redox groups on thesurface of the electrode. The measured value of the redox potential canthen be used to determine the concentration of an analyte, for examplean analyte in solution. In some embodiments, the solid state electrodeof the present invention has more than one redox-active species. In anembodiment, at least one redox-active species is sensitive to thepresence of an analyte (e.g., protons) and another redox-active speciesis insensitive to the presence of the analyte. Another aspect of theinvention relates to the measurement of the concentration of hydrogenion, or pH using the subject devices or systems. The surface modifiedsolid state sensors of the present invention can be used to measure thepH of a variety of solutions. The surface modified sensors of thepresent invention are robust, reliable, accurate, and/or can be madesuch that they do not require calibration.

In some embodiments, solid state electrodes are formed ofsemiconductors. Semiconductors have advantages as substrates and sensorsfor the methods of the present invention. Semiconductors have band gapsthat may be modified with the aid of chemical dopants, which can aid inpreparing sensitive and accurate sensors. In addition, semiconductorscan be less prone to fouling and degradation than other substrates.

Semiconductor surfaces, for example, inorganic semiconductors such assilicon and organic semiconductors, can be amenable to surfacemodification, e.g., covalent modification. Semiconductors generally haveelectronic band structures, the characteristics of which can bemodified, for example, by doping. In some cases, the semiconductor thatis used is silicon. An advantage of using silicon as a substrate and asan electrode is that silicon is amenable to mass production. Inparticular, semiconductor processing techniques are readily availablefor producing silicon electrodes in large quantities at low cost. Inaddition, existing semiconductor processing techniques make it feasibleto integrate electronic functionality into the material comprising thesilicon electrode. Another advantage of silicon is that it can formstrong covalent bonds, for example with carbon, nitrogen, oxygen, thusallowing for the facile and robust modification of the surface in amanner required for its intended uses. For instance, a silicon surfacecan be modified to attachment of any suitable redox-active agent.Silicon is also an advantageous surface for carrying out voltammetry inthat it is stable to a wide range of electrical potential withoutundergoing degradation.

An aspect of the invention is a surface modified solid state (e.g.,semiconductor) redox sensor that can measure analyte concentrationreliably and consistently with minimal intervention, such asre-calibration. Another aspect of the invention is a semiconductor redoxsensor that does not require calibration or re-calibration. The abilityto sense analytes, such as hydrogen ion without calibration has a numberof advantages for analyte measurements, for example, for in-linemonitoring. For example, it allows for ease of operator handling forsingle point measurements. In some embodiments, the sensors of thepresent invention are included in in-line operator-independent controlmeasurements. Such in-line measurements can be made independent of theoperator and can be used for process control, for example for pHmeasurements for process control. The subject sensors can be set toprovide real-time measurements of an analyte, including, but not limitedto real time-measurements of hydrogen ion concentration.

Semiconductor Substrates

An aspect of the invention provides an electrochemical sensor having aworking electrode formed of a semiconductor substrate. The semiconductorsubstrate can comprise any suitable semiconductor material includingthose known in the art and those described herein. The semiconductorsubstrate can be an inorganic semiconductor or an organic semiconductor.The semiconductor substrate can be doped or undoped. In some embodimentsthe semiconductor substrate comprises silicon.

A semiconductor substrate of the invention is generally a solid materialthat has electrical conductivity in between that of a conductor and thatof an insulator. The conductivity can vary over that wide range eitherpermanently or dynamically.

Inorganic semiconductor substrates of the invention can comprise, forexample, Group IV elemental semiconductors, such as diamond (C), silicon(Si), germanium (Ge); Group IV compound semiconductors, such as siliconcarbide (SiC), silicon nitride (SiN), silicon germanide (SiGe), GroupIII-V semiconductors, such as aluminum antimonide (AlSb), aluminumarsenide (AlAs), aluminum nitride (AlN), aluminum phosphide (AlP), boronnitride (BN), boron phosphide (BP), boron arsenide (BAs), galliumantimonide (GaSb), gallium arsenide (GaAs), gallium nitride (GaN),gallium phosphide (GaP), indium antimonide (InSb), indium arsenide(InAs), indium nitride (InN), indium phosphide (InP); Group III-Vternary semiconductor alloys, such as aluminum gallium arsenide (AlGaAs,AlxGa1-xAs), indium gallium arsenide (InGaAs, InxGa1-xAs), indiumgallium phosphide (InGaP), aluminum indium arsenide (AlInAs), aluminumindium antimonide (AlInSb), gallium arsenide nitride (GaAsN), galliumarsenide phosphide (GaAsP), aluminum gallium nitride (AlGaN), aluminumgallium phosphide (AlGaP), indium gallium nitride (InGaN), indiumarsenide antimonide (InAsSb), indium gallium antimonide (InGaSb), GroupIII-V quaternary semiconductor alloys, such as aluminum gallium indiumphosphide (AlGaInP, also InAlGaP, InGaAlP, AlInGaP), aluminum galliumarsenide phosphide (AlGaAsP), indium gallium arsenide phosphide(InGaAsP), Aluminum indium arsenide phosphide (AlInAsP), aluminumgallium arsenide nitride (AlGaAsN), indium gallium arsenide nitride(InGaAsN), indium aluminum arsenide nitride (InAlAsN), gallium arsenideantimonide nitride (GaAsSbN), Group III-V quinary semiconductor alloys,such as gallium indium nitride arsenide antimonide (GaInNAsSb), galliumindium arsenide antimonide phosphide (GaInAsSbP), II-VI semiconductors,cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telluride(CdTe), zinc oxide (ZnO), zinc selenide (ZnSe), zinc sulfide (ZnS), zinctelluride (ZnTe), Group II-VI ternary alloy semiconductors, such ascadmium zinc telluride (CdZnTe, CZT), mercury cadmium telluride(HgCdTe), mercury zinc telluride (HgZnTe), mercury zinc selenide(HgZnSe), Group I-VII semiconductors, such as cuprous chloride (CuCl),Group IV-VI semiconductors, such as lead selenide (PbSe), lead sulfide(PbS), lead telluride (PbTe), tin sulfide (SnS), tin telluride (SnTe),GroupIV-VI ternary semiconductors, such as lead tin telluride (PbSnTe),Thallium tin telluride (Tl2SnTe5), thallium germanium telluride(Tl2GeTe5), Group V-VI semiconductors, such as bismuth telluride(Bi2Te3), and Group II-V semiconductors, such as cadmium phosphide(Cd3P2), cadmium arsenide (Cd3As2), cadmium antimonide (Cd3Sb2), zincphosphide (Zn3P2), zinc arsenide (Zn3As2), and zinc antimonide (Zn3Sb2).

The inorganic semiconductor substrates of the invention can alsocomprise layered semiconductors, such as lead(II) iodide (PbI2),molybdenum disulfide (MoS2), gallium selenide (GaSe), tin sulfide (SnS),bismuth sulfide (Bi2S3), other semiconductors, such as copper indiumgallium selenide (CIGS), platinum silicide (PtSi), bismuth(III) iodide(BiI3), mercury(II) iodide (HgI2), thallium(I) bromide (TlBr), andmiscellaneous oxides, such as titanium dioxide: anatase (TiO2),copper(I) oxide (Cu2O), copper(II) oxide (CuO), uranium dioxide (UO2),and uranium trioxide (UO3).

In some embodiments of the invention, the semiconductor substrate cancomprise an organic semiconductor. The organic semiconductor is anysuitable organic material that has semiconductor properties. The organicsemiconductor substrates of the invention can comprise, for example,small molecules, short chain (oligomers) and long chain (polymers).Examples of semiconducting small molecules (e.g., unsaturated andaromatic hydrocarbons) are pentacene, anthracene, rubrene, tetracene,chrysene, pyrene, perylene, coronene, metal complexes of porphine andphthalocyanine, compounds such as zinc1,10,15,20-tetraphenyl-21H,23H-porphine, copper phthalocyanine, lutetiumbisphthalocyanine, and aluminum phthalocyanine chloride can be used.Suitable derivatives of these small molecules can also be used. In someembodiments, the organic semiconductor substrates of the invention cancomprise thin films.

Examples of semiconducting polymers or oligomers include suitableconjugated hydrocarbon or heterocyclic polymers or oligomers. Suitablepolymers or oligomers include: polyaniline, polypyrrole, andpolythiophene, poly(3-hexylthiophene), poly(p-phenylene vinylene), F8BT,polyacetylene, polydiacetylene, polyacene, polyphenylene, poly(phenylenevinylene), polyfuran, polypyridine, poly(thienylene vinylene),poly(ferrocenyl vinylene phenylene vinylene), poly(fluorine), andpoly(carbazole), and combinations thereof. Derivatives of thesepolymers, for instance derivatives having functional side chainsamenable to the attachment of redox active species, can be used.

Other examples of semiconducting polymers are poly(anilinesulfonicacid), poly(ferrocenyl vinylene phenylene vinylene), poly(fluorine), andpoly(carbazole). The organic semiconductor substrates of the inventioncan comprise, for example, organic charge-transfer complexes, and“linear backbone” polymers derived from polyacetylene, such aspolyacetylene itself, polypyrrole, and polyaniline. Charge-transfercomplexes can exhibit similar conduction mechanisms to inorganicsemiconductors. This includes the presence of a hole and electronconduction layer and a band gap. The materials can exhibit tunneling,localized states, mobility gaps, and phonon-assisted hopping. Organicsemiconductors can be doped. In some embodiments, the invention canutilize highly doped organic semiconductors, for example Polyaniline(Ormecon) and PEDOT:PSS. In some cases organic semiconductors can beproduced such that they are transparent and/or flexible, which can beuseful in some embodiments.

The semiconductors of the invention can typically be characterized ashaving a band gap, the band gap representing the amount of energyseparating the valence and conduction bands of the semiconductor. Theaddition of dopants makes the band gap smaller, tending to allow morefacile promotion of electrons from the valence band to the conductionband. A smaller band gap can result in higher conductivity for thesemiconductor substrate. The band gap and conductivity characteristicsof the semiconductor substrates can be controlled in some cases by theintroduction of dopants. In some cases upon the addition of asufficiently large proportion of dopants, the semiconductor substratesof the invention can conduct electricity nearly as well as metals.Depending on the kind of dopant or impurity, a doped region ofsemiconductor can have more electrons or holes, and is named N-type orP-type (herein also “n-type” and “p-type”) semiconductor material,respectively. Junctions between regions of N- and P-type semiconductorscreate electric fields, which cause electrons and holes to be availableto move away from them, and this effect is critical to semiconductordevice operation. Also, a density difference in the amount of impuritiesproduces a small electric field in the region which is used toaccelerate non-equilibrium electrons or holes.

In some embodiments the presence of the band gap can be advantageous.For example, when performing electrochemistry, when the Fermi energy ofa doped semiconductor lies at the same energy as the solution/molecularredox potential at a certain potential, generally no net transfer ofcharge/current will flow from the redox species (immobilized on thesurface or in the solution) to the substrate or from the substrate tothe redox species. This potential is sometimes referred to as theflatband potential. The location of the flatband potential can beinfluenced by dopant densities. The Mott-Schottky equation (see P.Schmuki, H. Bohni and J. A. Bardwell, J. Electrochem. Soc., 1995, 142,1705) can be used to estimate the flat band potential.

A conductor electrode generally does not have a flatband potential thushas a broad potential window where current can flow from the redoxspecies to the substrate. For example, when the conductor electrode isexposed to an aqueous solution containing a mixture of several redoxactive species, currents corresponding to these redox species (i.e.,non-specific adsorption) may be recorded unless additional efforts aremade to screen off these non-specific interactions, e.g., putting adiluent layer onto the conductor electrode. The non-specificinteractions can be reduced or eliminated by using a semiconductorelectrode wherein the semiconductor has a the band gap that has alimited potential window, such that the semiconductor can only conductcurrent for the electrochemical reaction occurring within that limitedwindow. For example, the limited window can be between −1.0 to 0 V.Thus, in some embodiments, non-specific interactions in the solution canbe reduced or eliminated by using a semiconductor electrode of theinvention with the appropriate dopant density (which can be estimatedusing the Mott-Schottky equation).

One useful aspect of the semiconductors of the invention is that theirconductivity can be modified by introducing impurities (dopants) intotheir crystalline lattice or amorphous regions. The process of addingcontrolled impurities to a semiconductor can be referred to as doping.The amount of impurity, or dopant, added to an intrinsic (pure)semiconductor alters its level of conductivity. Doped semiconductors maybe referred to as extrinsic. The semiconductors of the present inventioncan be either intrinsic or extrinsic semiconductors.

Suitable dopants can be chosen, as is known in the art, on the atomicproperties of the dopant and the material to be doped. In general,dopants that produce the desired controlled changes are classified aseither electron acceptors or donors. A donor atom that activates (e.g.,becomes incorporated into the crystal lattice) donates weakly-boundvalence electrons to the material, creating excess negative chargecarriers. These weakly-bound electrons can move about in thesemiconductor relatively freely and thus can facilitate electricalconduction in the presence of an electric field. In some cases, thedonor atoms introduce some states below, but very close to, theconduction band edge. Electrons at these states can be thermally excitedto conduction band, becoming free electrons, in some cases, at roomtemperature. In some embodiments an activated acceptor dopant isutilized. The activated acceptor can produce a hole. Semiconductorsdoped with donor impurities are typically called N-type, while thosedoped with acceptor impurities are typically known as P-type. In someembodiments of the invention, the semiconductor of the invention canhave both donor and acceptor dopants. In some embodiments thesemiconductor can have both n type and p type charge carriers. The n orp type designation generally indicates which charge carrier acts as thematerial's majority carrier. The opposite carrier is generally calledthe minority carrier, which, in some cases, exists due to thermalexcitation at a lower concentration than the majority carrier.

As described herein, where the semiconductor is a Group IV semiconductorsuch as silicon or germanium, suitable electron donors include, forexample, phosphorous, arsenic, antimony, and bismuth. In someembodiments the dopant is phosphorous. In some embodiments the dopant isantimony. Where the semiconductor is a Group IV semiconductor such assilicon or germanium, suitable electron acceptors include boron,aluminum, gallium, and the like. In some embodiments, the dopant isboron. Where the semiconductor comprises an element in another groupthan Group IV, as is known in the art, elements that are outside of thatgroup can act as either n type of p type dopants.

In addition to modification through doping, the resistance ofsemiconductors can in some cases, for example, be modified dynamicallyby applying electric fields. The ability to controlresistance/conductivity in semiconductor substrate or within regions ofsemiconductor substrate dynamically through the application of electricfields can be useful in some embodiments.

The semiconductor substrate can be in any form that is amenable to theproduction of a semiconductor electrode. The semiconductor substrate cancomprise a monolithic piece of the semiconductor, a coating of thesemiconductor deposited onto another material, or a powder ofsemiconductor particles. The semiconductor substrate can be a monolithicform such as a chip, wafer, rod, needle, block, ingot, or the like. Thesemiconductor substrate can alternately be in particulate form, forexample in the form of powder comprised of particles. The particles canbe of arbitrary shape or can be in the form of fibers, sheets, beads,discs, or balls. Where the substrate is in the form of a powder made upof particles, it will generally be formed into a composite electrode asdescribed in more detail below.

The semiconductor substrate of the present invention can be a thin layerof semiconductor that is formed upon another material, for example athin layer of semiconductor formed on glass would constitute asemiconductor substrate.

In an embodiment, the semiconductor substrate can include a layer ofsemiconductor material having a thickness of about 0.1 nanometers (“nm”)and 5000 nm, or between about 1 nm and 1000 nm, or between about 10 nmand 500 nm. In another embodiment, the semiconductor substrate caninclude a layer of silicon having a thickness of about 0.1 nanometers(“nm”) and 5000 nm, or between about 1 nm and 1000 nm, or between about10 nm and 500 nm.

In some embodiments the semiconductor substrate used to make theelectrode is a composite material comprising semiconductor particlesdispersed in a matrix or binder. The semiconductor substrate can be madeof a composite material comprising a powder of semiconductor dispersedin a binder to make a composite semiconductor substrate. Thesemiconductor powder can be in the form of spheres, crystallites, rods,fibers, or any other arbitrary shape. In an embodiment the compositeelectrode is made of semiconductor crystallites dispersed in a polymericmatrix. The matrix or binder can be an organic, inorganic, ororganometallic polymer. Non-limiting examples of useful inorganicpolymeric materials include polyphosphazenes, polysilanes, polysiloxane,polygermanes, polymeric sulfur, polymeric selenium, silicones, andmixtures of any of the foregoing.

In some embodiments the polymer can be an organic polymer. Non-limitingexamples of suitable organic polymeric materials include, but are notlimited to, thermoset materials and thermoplastic materials.Non-limiting examples of polymers useful in the invention includepolyesters such as polyethylene terephthalate, polybutyleneterephthalate, and polyethylene naphthalate, polycarbonates, polyolefinssuch as polyethylene, polypropylene, and polyisobutene, acrylic polymerssuch as copolymers of styrene and an acrylic acid monomer, and polymerscontaining methacrylate, polyamides, thermoplastic polyurethanes, vinylpolymers, polyimides, polyamides, polytetrafluoroethelene and otherfluoropolymers, and mixtures of any of the foregoing.

The binder can be insulating, semiconductive, or conductive. In anembodiment, the binder is a material, such as a polymer, which is aninsulating material. Where an insulating binder is used, the currentwill tend to only flow through the dispersed semiconductor powder. Insome embodiments, the binder includes conductive components. In someembodiments, the binder comprises a conductive polymer such aspolyaniline, polyacetylene, poly(alkylthiophene), poly(alkylpyrrole),and the like. In some embodiments, the conductive component can compriseconductive particles such as metal particles, such as nickel particlesother conductive particles including carbon particles. In someembodiments, the conductive component is chosen such that the conductivecomponent such as the conductive polymer exhibits reduction and/oroxidation potentials that are outside of the reduction and/or oxidationpotentials of the redox active moieties.

The composite semiconductor substrate can be formed by mixing thesemiconductor powder with a monomer, oligomer, or prepolymer and curingthe monomer, oligomer or prepolymer to form a polymeric matrix. Thepolymerization can be initiated in any manner known in the art ordisclosed herein. The polymerization can be initiated, for example,thermally or photochemically in the presence of an initiator. Thepolymerization can be carried out with one or more crosslinkers. Thecross-linkers can be chosen to adjust the physical properties of thepolymeric matrix and thus adjust the properties of the compositesemiconductor substrate. The composite semiconductor substrate can beformed by mixing the semiconductor powder with a molten thermoplasticpolymer, forming the substrate, and allowing the mixture to cool. Thecomposite semiconductor substrate can be formed by mixing thesemiconductor powder with a polymer or prepolymer in a solvent, andallowing the solvent to evaporate to form the composite. Combinations ofany of the above methods can be used.

The electrical properties of the composite semiconductor substrate canbe affected by the amount of semiconductor, the particle size, and theparticle shape. In general, the amount of semiconductor in the compositeis high enough to create conductive pathways throughout the material.This amount of material necessary to provide conductive paths across thematerial is sometimes called the percolation threshold. The amount ofsemiconductor particles for conductivity can also depend on theprocessing conditions such as the viscosity of the binder and the amountof mixing. The amount of semiconductor is generally set at a level atwhich the physical properties of the material, such as mechanicalstrength and flexibility will not suffer to the point that the materialis not useful. The amount of semiconductor will generally be from 0.1volume percent to 70 volume percent of the composite material. In someembodiments the amount of semiconductor may be from 1 volume percent to50 volume percent. In some embodiments the amount of semiconductor maybe from 10 volume percent to 40 volume percent. The amount ofsemiconductor can be from 1% to 5%, 5% to 10%, 10% to 15%, 10% to 20%,20% to 30%, 30% to 40%, 40% to 50% or 50% to 60%.

The composite semiconductor substrate can be formed by methods used forshaping polymeric materials such as coating, molding, and casting intoshapes that are useful as electrodes. The composite semiconductorsubstrate electrode will generally be connected to an electricallyconductive wire to apply current and potential. The material can becast, coated, and/or molded onto a conductive substrate such as a metalto form a conductive junction for connecting conductors for transfer ofcurrent to and from the composite electrode.

The semiconductor substrates of the invention generally have a highenough electrically conductivity to act as electrodes, to transmitcurrent for the oxidation and/or reduction of the bound redox-activemoieties. To make the semiconductor substrate more conductive, thesemiconductor substrate can include impurities or dopants to increaseelectrical conductivity and reduce the resistivity.

The electrical resistivity (reciprocal of conductivity) can be forexample 0.1 (ohm-centimeters), 1 (ohm-centimeters), 10(ohm-centimeters), 100 (ohm-centimeters), to in excess of 1000 or even10,000 (ohm-centimeters) or even higher which is comparable to graphiteand conventional metallic conductors.

In some embodiments the resistivity of the semiconductor substrate isthe range of 0.0001 to 100,000 Ω-cm (or ohm-centimeters). In someembodiments the resistivity of the semiconductor substrate is the rangeof 0.001 to 10,000 Ω-cm. In some embodiments the resistivity of thesemiconductor substrate is the range of 0.01 to 1000 Ω-cm. In someembodiments the resistivity of the semiconductor substrate is the rangeof 0.1 to 100 Ω-cm. In some embodiments the resistivity of thesemiconductor substrate is within the range of 1 to 100 Ω-cm. In someembodiments the resistivity of the semiconductor substrate is within therange of 10 to 90 Ω-cm. In some embodiments the semiconductor substrateis single crystal semiconductor is Si(100) that is P-type with aresistivity of 10 to 90 Ω-cm. In some cases several semiconductorsubstrates with different resistivities may be used. For example, asystem of the invention may comprise one lightly doped semiconductorsubstrate having one redox active species bound to it, and also a morehighly doped semiconductor surface having another redox active speciesbound to it. For example, a system of the invention may comprise onelightly doped semiconductor surface having a pH sensitive redox activemoiety such as anthraquinone bound thereto, and a second semiconductorsurface that is more highly doped having a hydrogen ion insensitiveredox active moiety such as ferrocene bound to it.

In some cases different regions of the semiconductor substrate can bedoped at different levels. For example, it is well known in thesemiconductor processing art that a mask can be used to cover someregions of the semiconductor, while leaving other regions exposed. Theexposed regions can be treated selectively with dopants resulting in asemiconductor surface wherein some regions are doped differently thanother regions. By using multiple steps with various masks, theconductivity properties of different regions of the semiconductorsurface can be controlled. Thus, some regions can have highconductivity, some low, some regions can have a large band gap, andother regions can have small band gaps, some regions can have N-doping,some P-doping, and some no doping. In addition, the various regions canbe connected with conducting regions, for example deposited metal, e.g.,gold in to be able to electrically address the various regions.

Silicon Substrates

Another aspect of the invention provides an electrochemical sensorhaving a semiconductor substrate that includes silicon. The siliconsubstrate can have a surface onto which are attached redox-activemoieties. The silicon substrate can comprise amorphous silicon orsilicon comprising a variety of crystalline forms. The silicon substratecan also be polycrystalline. In some embodiments the silicon substratecan have both amorphous and crystalline regions. In some embodiments,the silicon can be nanocrystalline or microcrystalline silicon.Nanocrystalline silicon and microcrystalline silicon can be used todescribe an allotropic form of silicon with paracrystalline structurehaving small grains of crystalline silicon within the amorphous phase.Where the silicon substrate is crystalline, the surface of the siliconsubstrate can have various crystalline faces on the surface. Crystallinesilicon is generally in a face centered cubic (fcc) form. In someembodiments, such as where a polycrystalline silicon is used, thesurface may have multiple crystalline planes exposed. Where singlecrystal silicon is used, in some cases, the silicon substrate can bemade to have one or more crystal planes represented or predominantlyrepresented on the surface. In some embodiments, the surface of thesilicon substrate will comprise one or more crystal planes having acrystalline lattice of (xxx) wherein x is an integer corresponding tothe lattice defining the crystal plane. In some embodiments the crystalplanes (100), (010), (001), (110), (101), or (112) may be predominantlyrepresented at the surface. In some embodiments a silicon substrate hasthe (100) plane predominantly represented at the surface.

The silicon electrode of the present invention can comprise a polishedor an unpolished silicon substrate. Silicon is generally polished priorto silicon processing, for example, building features such astransistors. In some embodiments, such as those embodiments whereelectronic functionality is incorporated into a silicon sensor, apolished silicon surface may be desirable. In other embodiments, anunpolished silicon substrate can be used. An unpolished siliconsubstrate can be less expensive than a silicon substrate that has gonethrough a polishing step. In addition, an unpolished silicon substratecan have a higher surface area for a given area of silicon than apolished silicon substrate.

The silicon electrode of the present invention can comprise poroussilicon. An advantage of porous silicon is an increase of the effectivesurface area. An increased surface area can be advantageous forproviding a higher signal from the oxidation and reduction of thesurface bound redox moieties due to a higher number of such moieties incontact with the sample. As is known in the art, if the surface is tooporous, it can become less robust. Therefore the level of porosity canbe controlled to maximize important properties for the particularapplications. The porous silicon can be prepared by, for example,galvanostatic, chemical, or photochemical etches from silicon wafers. Insome embodiments, chemical etching with hydrofluoric acid (HF) can beused to produce a porous silicon substrate. In some embodiments, theaverage pore size of the silicon substrate ranges from 1 nm to 500 nm.Pore size can be measured by, for example, nitrogen gas adsorption or Hgporosimetry. In some embodiments, the amount of porosity ranges between1% and 98%. In some embodiments, the amount of porosity ranges between5% and 75%. In some embodiments, the amount of porosity ranges between10% and 50%. In some embodiments, the amount of porosity ranges between20% and 40%. In some embodiments the porosity is between 1% to 5%, 5% to10%, 10% to 30%, 20% to 40%, 30% to 50%, or 40% to 60%. The porositymeasurement can be made on an area percent basis or a volume percentbasis.

In addition, porous silicon could be readily integrated with existingsilicon-based integrated circuit (IC) manufacturing processes.

The silicon substrate can be in any form that is amenable to theproduction of a silicon electrode. The silicon substrate can comprise amonolithic piece of silicon, a coating of silicon deposited onto anothermaterial, or a powder of silicon particles. The silicon substrate can bea monolithic form such as a chip, wafer, rod, needle, block, ingot, orthe like. The silicon substrate can alternately be in particulate form,for example in the form of powder comprised of particles. The particlescan be of arbitrary shape or can be in the form of fibers, sheets,beads, discs, polyhedra, or balls. Where the substrate is in the form ofa powder made up of particles, it will generally be formed into acomposite electrode as described in more detail below.

In some embodiments the semiconductor electrode is made from singlecrystal silicon. The single crystal silicon can be made by zone melting,also called zone refining, a process in which rods of metallurgicalgrade silicon are first heated to melt at one end. Then, the heater istypically slowly moved down the length of the rod, keeping a smalllength of the rod molten as the silicon cools and re-solidifies behindit. Since most impurities tend to remain in the molten region ratherthan re-solidify, when the process is complete, most of the impuritiesin the rod will typically have been moved into the end that was the lastto be melted. This end is then cut off and discarded, and the processrepeated if a still higher purity is desired. The single crystal siliconof the invention can also be produced via the Czochralski process,(CZ-Si) which tends to be inexpensive and is capable of producing largesize crystals.

In some embodiments the silicon electrode is polycrystalline. As usedherein, the term “polysilicon” is used interchangeably with the term“polycrystalline silicon”. In some embodiments, the polysilicon isdeposited. The polycrystalline silicon can be deposited by low pressurechemical vapor deposition (LPCVD), plasma-enhanced chemical vapordeposition (PECVD), or solid-phase crystallization (SPC) of amorphoussilicon in certain processing regimes These processes can requirerelatively high temperatures, for example, above 300° C. Thepolycrystalline silicon electrodes can also be made, for example onpolymeric substrates, using laser crystallization to crystallize aprecursor amorphous silicon (a-Si) material on a plastic substratewithout melting or damaging the plastic. In some cases, the for example,short, high-intensity ultraviolet laser pulses are used to heat thedeposited a-Si material to above the melting point of silicon, withoutmelting the entire substrate. By controlling the temperature gradients,the crystal size on the electrodes can be controlled. Grain sizes canbe, for instance from 10 nanometer to 1 micrometer. Another method toproduce polysilicon at low temperatures for the electrodes of thepresent invention is a metal-induced crystallization in which anamorphous silicon thin film is crystallized, for example at temperaturesat or above 150° C., while in contact of a metal film such as aluminum,gold, or silver. The polycrystalline silicon electrodes can also beformed onto a metal structure such as a wire. For example, the end of acylindrical wire can be coated with polysilicon, which can bederivatized with redox active species as described herein. The structurecan be used as an electrode or portion of an electrode with siliconportion accessible to the medium containing the analyte, and the wireacting to connect the silicon electrode to the parts of the systemproviding voltage and allowing for the flow of current.

An advantage of polysilicon over amorphous silicon (a-Si) is that themobility of the charge carriers can be orders of magnitude larger thanin single crystal silicon and the material also can show greaterstability under electric field and light-induced stress.

The silicon substrate of the present invention can be a thin layer ofsilicon that is formed upon another material, for example a thin layerof silicon formed on glass would constitute a silicon substrate.

In some embodiments the silicon substrate used to make the electrode isa composite material comprising silicon particles dispersed in a matrixor binder. The silicon substrate can be made of a composite materialcomprising a powder of silicon dispersed in a binder to make a compositesilicon substrate. The silicon powder can be in the form of spheres,crystallites, rods, fibers, or any other arbitrary shape. In anembodiment the composite electrode is made of silicon crystallitesdispersed in a polymeric matrix. The matrix or binder can be an organic,inorganic, or organometallic polymer. Non-limiting examples of usefulinorganic polymeric materials include polyphosphazenes, polysilanes,polysiloxane, polygermanes, polymeric sulfur, polymeric selenium,silicones, and mixtures of any of the foregoing.

In some embodiments the polymer can be an organic polymer. Non-limitingexamples of suitable organic polymeric materials include, but are notlimited to, thermoset materials and thermoplastic materials.Non-limiting examples of polymers useful in the invention includepolyesters such as polyethylene terephthalate, polybutyleneterephthalate, and polyethylene naphthalate, polycarbonates, polyolefinssuch as polyethylene, polypropylene, and polyisobutene, acrylic polymerssuch as copolymers of styrene and an acrylic acid monomer, and polymerscontaining methacrylate, polyamides, thermoplastic polyurethanes, vinylpolymers, polyimides, polyamides, polytetrafluoroethelene and otherfluoropolymers, and mixtures of any of the foregoing.

The binder can be insulating, semiconductive, or conductive. In anembodiment, the binder is a material, such as a polymer, that is aninsulating material. Where an insulating binder is used, the currentwill tend to only flow through the dispersed silicon powder. In someembodiments, the binder includes conductive components. In someembodiments, the binder comprises a conductive polymer such aspolyaniline, polyacetylene, poly(alkylthiophene), poly(alkylpyrrole),and the like. In some embodiments, the conductive component can compriseconductive particles such as metal particles, such as nickel particlesother conductive particles including carbon particles. In someembodiments, the conductive component is chosen such that the conductivecomponent such as the conductive polymer exhibits reduction and/oroxidation potentials that are outside of the reduction and/or oxidationpotentials of the redox active moieties.

The composite silicon substrate can be formed by mixing the siliconpowder with a monomer, oligomer, or prepolymer and curing the monomer,oligomer or prepolymer to form a polymeric matrix. The polymerizationcan be initiated in any manner known in the art or disclosed herein. Thepolymerization can be initiated, for example, thermally orphotochemically in the presence of an initiator. The polymerization canbe carried out with one or more crosslinkers. The cross-linkers can bechosen to adjust the physical properties of the polymeric matrix andthus adjust the properties of the composite silicon substrate. Thecomposite silicon substrate can be formed by mixing the silicon powderwith a molten thermoplastic polymer, forming the substrate, and allowingthe mixture to cool. The composite silicon substrate can be formed bymixing the silicon powder with a polymer or prepolymer in a solvent, andallowing the solvent to evaporate to form the composite. Combinations ofany of the above methods can be used.

The electrical properties of the composite silicon substrate can beaffected by the amount of silicon, the particle size, and the particleshape. In general, the amount of silicon in the composite is high enoughto create conductive pathways throughout the material. This amount ofmaterial necessary to provide conductive paths across the material issometimes called the percolation threshold. The amount of siliconparticles required for conduction can also depend on the processingconditions such as the viscosity of the binder and the amount of mixing.The amount of silicon is generally set at level at which the physicalproperties of the material, such as mechanical strength and flexibilitywill not suffer to the point that the material is not useful. The amountof silicon will generally be from 0.1 volume percent to 70 volumepercent of the composite material. In some embodiments the amount ofsilicon may be from 1 volume percent to 50 volume percent. In someembodiments the amount of silicon may be from 10 volume percent to 40volume percent. The amount of silicon can be from 1% to 5%, 5% to 10%,10% to 15%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or 50% to60%.

The composite silicon substrate can be formed by methods used forshaping polymeric materials such as coating, molding, and casting intoshapes that are useful as electrodes. The composite silicon substrateelectrode will generally be connected to an electrically conductive wireto apply current and potential. The material can be cast, coated, and/ormolded onto a conductive substrate such as a metal to form a conductivejunction for connecting conductors for transfer of current to and fromthe composite electrode.

The silicon substrates of the invention generally have a high enoughelectrically conductivity to act as electrodes, and to transmit currentfor the oxidation and/or reduction of the bound redox-active moieties.To make the silicon substrate more conductive, the silicon substratewill generally include impurities or dopants to increase electricalconductivity. Where polycrystalline silicon is used, the polycrystallinesilicon electrode can either be deposited as doped polycrystallinesilicon (in situ doped) or can be deposited undoped and subsequentlydoped with an impurity dopant such as phosphorus or boron by ionimplantation or a thermal diffusion process. Dopant impurities, such asphosphorus and boron, tend to diffuse much more rapidly along the grainboundaries than they do through the silicon itself.

The dopant for a type IV semiconductor such as silicon, can be, forexample, either an electron donor or an electron acceptor. Suitableelectron donors are phosphorous, arsenic, antimony, and bismuth. In someembodiments the dopant is phosphorous. In some embodiments the dopant isantimony. Suitable electron acceptors are boron, aluminum, gallium, andthe like. In some embodiments, the dopant is boron. In some embodiments,electron acceptors can impart a chemical resistance to the siliconelectrode.

Where the silicon substrate is a monolithic material such as a wafer,the dopant can be either distributed throughout the bulk of the silicon,or can be limited to the surface region of the silicon wafer. In someembodiments, for example where the silicon substrate comprises multiplezones with different redox active moieties, the dopant can bedistributed such that isolated regions of the surface of the siliconsubstrate are conductive.

The dopant is generally present in an amount greater than 0.01 weightpercent of the silicon, and generally in an excess of 0.1 percent of thesilicon. Generally, the dopant is less than 3 weight percent of thesilicon, and almost always less than 6 weight percent of the silicon.The presence of small amounts of the dopant can increase the electricalconductivity. The dopant may not be homogenously distributed throughoutthe silicon, and the local concentration may vary between differentregions of the silicon material.

The electrical resistivity (reciprocal of conductivity) can be forexample 0.1 (ohm-centimeters), 1 (ohm-centimeters), 10(ohm-centimeters), 100 (ohm-centimeters), to in excess of 1000 or even10,000 (ohm-centimeters) or even higher which is comparable to graphiteand conventional metallic conductors.

In some embodiments the resistivity of the silicon substrate is therange of 0.0001 to 100,000 a-cm. In some embodiments the resistivity ofthe silicon substrate is the range of 0.001 to 10,000 Ω-cm. In someembodiments the resistivity of the silicon substrate is the range of0.01 to 1000 Ω-cm. In some embodiments the resistivity of the siliconsubstrate is the range of 0.1 to 100 Ω-cm. In some embodiments theresistivity of the silicon substrate is within the range of 1 to 100Ω-cm. In some embodiments the resistivity of the silicon substrate iswithin the range of 10 to 90 Ω-cm. In some embodiments the siliconsubstrate is single crystal silicon is Si(100) that is P-type with aresistivity of 10 to 90 Ω-cm. In some cases several silicon substrateswith different resistivities may be used. For example, a system of theinvention may comprise a one lightly doped silicon substrate having oneredox active species bound to it, and also a more highly doped siliconsurface having another redox active species bound to it. For example, asystem of the invention may comprise one lightly doped silicon surfacehaving a pH sensitive redox active moiety such as anthraquinone boundthereto, and a second silicon surface that is more highly doped having ahydrogen ion insensitive redox active moiety such as ferrocene bound toit.

In some cases different regions of the silicon substrate can be doped atdifferent levels. For example, it is well known in the semiconductorprocessing art that a mask can be used to cover some regions of thesilicon, while leaving other regions exposed. The exposed regions can betreated selectively with dopants resulting in a silicon surface whereinsome regions are doped differently than other regions. By using multiplesteps with various masks, the conductivity properties of differentregions of the silicon surface can be controlled. Thus, some regions canhave high conductivity, some low, some regions can have a large bandgap, and other regions can have small band gaps, some regions can haveN-doping, some P-doping, and some no doping. In addition, the variousregions can be connected with conducting regions, for example depositedmetal, e.g., gold to be able to electrically address the variousregions.

In some embodiments, Nisil is used. Nisil is an alloy of nickel andsilicon. In some embodiments Nisil with 4%-5% silicon is used. In someembodiments Nisil with 4.4% silicon is used. In some embodimentsNicrosil is used. Nicrosil is a nickel alloy. In some cases Nicrosilcomprising 14.4% chromium, 1.4% silicon, and 0.1% magnesium is used.

In some cases, for example, where the silicon substrate is cast silicon,the silicon substrate will include, for example, a silicide of atransition metal to provide castability. The silicide of the transitionmetal can provide favorable mechanical properties to the cast alloy.Typical metals useful in providing the transition metal silicide presentin the silicon electrode of the secondary cell of this invention includetitanium, zirconium, hafnium, vanadium, columbium, chromium, molybdenum,tungsten, manganese, iron, cobalt, nickel, copper, ruthenium, rhodium,palladium, osmium, iridium, platinum, gold, and silver. Most commonly,the transition metal present as the silicide in the silicon alloy may bea silicide of manganese, chromium, iron, cobalt, nickel, or molybdenum.The amount of the silicide may be sufficient to provide satisfactorycastability but not great enough to deleteriously effect the propertiesof the silicon, i.e., from 2 percent or more up to as high as 30 percenttransition metal, elemental basis.

Redox-Active Moieties

In some embodiments, an electrochemical sensor comprises a solid state(e.g., semiconductor) surface that is modified with redox-activefunctional groups. At least one redox-active functional group on thesurface is sensitive to the presence and or the level of a substance inthe solution. In some embodiments, the semiconductor surface can have atleast one redox-active functional group sensitive to an analyte, and atleast one redox-active functional group that is substantiallyinsensitive to the analyte to be tested. When used in this manner, thesubstantially insensitive group can act as a reference, allowing forgreater accuracy and reproducibility of the measurements.

In some situations, an electrochemical sensor can include a layer of anitride, such as silicon nitride, having immobilized thereon aredox-active moiety that is sensitive to the presence of an analyte,such as H+, and/or a redox-active moiety that is insensitive to thepresence of the analyte.

In some situations, redox active moieties that are sensitive and/orinsensitive to the presence or absence of an analyte are bound to asurface of a solid state working electrode, such as a semiconductor(e.g., silicon) surface, through a surface-to-carbon interaction, suchas, e.g., a silicon-to-carbon bond in cases in which the workingelectrode is formed of silicon. The silicon-to-carbon interaction can bea covalent interaction. The carbon atom in such a case is a carbon atomof a redox active moiety. In other situations, redox active moieties canbe bound to a surface of a working electrode through surface-to-oxygen,surface-to-sulfur, and/or surface-to-carbon interactions, which can becovalent interactions.

The redox groups can be chemically or physically bound to the surface.The redox groups can be attached to the semiconductor covalently (e.g.,via Si—C, Si—O, or Si—S bonds), can be adsorbed to the semiconductor, orcan be attached to polymers that are either covalently or non-covalentlybound to the surface. Covalent binding of either the redox group or thepolymer to which the redox group is a part can be beneficial inimproving the lifetime and stability of the electrode. Semiconductormaterials such as silicon and germanium can form covalent bonds withcarbon, and thus is a desirable substrate for functionalizing withcarbon based molecules. The covalent binding to the surface can bethrough a bond between the semiconductor, e.g., silicon, and carbon,oxygen, nitrogen, sulfur, or other atom. In some embodiments the bond isbetween silicon and carbon. In some embodiments the bond is betweensilicon and oxygen. The physical bonding can occur through adsorption,and can include, for example, spontaneous self assembly onto thesemiconductor surface of molecules such as those derived from fattyacids which comprise redox active moieties.

Where a linker group is used, the linker can be small, for example oneto 3 atoms, or can be longer, e.g., 20 to 100 atoms, or can be any sizebetween large and a small linker. Where a short linker is used, theredox-active moiety is held close to the surface. Where a longer groupis used, the redox active moiety may be able to move away from thesurface, for example into the solution. Linker groups can comprisehydrophilic, hydrophobic groups, or mixtures thereof. Linker groups cancomprise, for example, hydrocarbons, esters, ethers, amides, amines,carbonyls, thiols, olefins, silicones, or other organic, inorganic ororganometallic groups. The linker groups can be formed by polymerizationor oligomerization reactions such as free radical, cationic, or anionicpolymerization. The linker group can comprise, for example, ethyleneoxide, propylene oxide, vinyl ether, or acrylamide repeat units. Linkerscan have ring structures including aromatic rings. The variation in thelinker structure can be used to vary the mobility of the redox-activemoiety in the solution.

As used herein, the term moiety generally refers to a portion of amolecule or substituent. A redox-active moiety may be highlysubstituted, and can still act as a redox-active moiety. As used herein,the terms “redox active moiety”, “redox active group”, “redox activefunctional group”, and “redox group” are used interchangeably. Thus, forexample, the redox-active moiety ferrocene includes substitutedferrocenes, ferrocene polymers, and ferrocene covalently attached to thesurface with or without linker molecules.

In some embodiments, the redox-active moiety can be incorporated into apolymer, and the polymer comprising the redox active moiety can beimmobilized onto the semiconductor surface. The immobilization of thepolymer can be either chemical or physical. The immobilization of thepolymer can be through covalent bonds, or through adsorption of thepolymer to the semiconductor surface.

In some embodiments, the redox-active moiety is bound to a particle thatis bound to semiconductor. The particle is generally an electricallyconductive particle. The particle attached to the semiconductor surfacein a manner that allow for current to flow between the semiconductorsurface and the particle. The particles can be attached chemically orphysically to the surface. For example, the redox-active moiety can beattached to a carbon particle, and the carbon particle attached to thesemiconductor surface. In some embodiments, the carbon particle can be acarbon nanotube. In some embodiments of the invention, carbon nanotubescan be attached to the surface of the semiconductor, where there areredox-active groups attached to the carbon nanotubes. For instance,attachment of well-aligned single-walled carbon nanotubes architectureto a single-crystal silicon surface can be used. In some embodiments,for example, ferrocenemethanol molecules are attached to single walledcarbon nanotube (SWCNT) arrays that are directly anchored to the siliconsurface, for example, a (100) surface. For example, single wall carbonnanotubes can be coupled to the surface using this method as describedin Yu et al., Electrochimica Acta 52 (2007) 6206-6211.

The redox-active moieties generally have reversible redox activity withwell-defined cyclic voltammetry oxidation and/or reduction peaks. Asuitable reference redox reagent can vary from application toapplication or medium to medium depending on the intended use.

The position of the reduction and/or oxidation potentials of the redoxactive moiety can be chosen to improve the accuracy and quality of themeasurement of redox potential. In some cases, the reduction and oroxidation potential can be chosen to be away from other redox activespecies. The silicon surface, for example, generally has a wide windowin which to perform measurement of reduction or oxidation potentialwithout interfering with the measurement of the reduction and oroxidation of the redox active moieties bound to the surface. The siliconsurface can generally be used to measure oxidation and/or reductionpotentials from between negative 2 V to positive 2 V. In some cases, forexample where the medium is an aqueous medium, the reduction and/oroxidation potential of the redox-active moiety can be chosen so as notto fall within the reduction or oxidation potential of the medium tominimize interference. This can be useful where cyclic voltammetry isused, and is less important when square wave voltammetry is used.

Redox-active moieties that are insensitive to the presence of analytesshould show little or no change in their oxidation and/or reductionpotentials in the presence or absence of such analytes. Redox-activemoieties that are generally insensitive to the presence of analytes, andin particular are insensitive to the presence of hydrogen ion include:ferrocene, polyvinylferrocene, Os(bpy)₂Cl₂, Ru(bpy)₂Cl₂, viologen,polyviologen, and polythiophene. Redox reagents having a high degree ofelectrochemical reversibility are generally preferred. FIG. 5 showsexamples of the hydrogen ion insensitive redox-active moiety ferrocenebound to a silicon surface.

Non-limiting redox-active moieties that are sensitive to hydrogen ioninclude: quinones, anthroquinones, phenanthroquinones, phenylenediamines, catechols, phenothiazinium dyes, and monoquaternizedN-alkyl-4,4′-bipyridinium. In some embodiments the redox-active moietythat is sensitive to the presence of hydrogen ion can include inorganicmaterials and metal oxides. Hydrogen ion sensitive inorganicredox-active inorganic moieties include Prussian Blue, Ni(OH)₂, andRuO_(x). FIG. 6 shows examples of the hydrogen ion sensitiveredox-active moiety anthracene covalently bound to a silicon surface.FIG. 7 shows an example of a silicon surface having covalently boundthereto both the hydrogen ion sensitive redox-active moiety ferroceneand the hydrogen ion insensitive redox-active agent anthracene.

In some embodiments the analyte is carbon monoxide (CO). An example of aCO sensitive redox-active agent is ferrocenyl ferrazetine disulfide orferrazetine-ferrazetine disulfide. A CO insensitive redox-active agentcan be, for example, ferrocene.

In some embodiments the analyte is an alkali metal. Alkali metalsensitive redox-active agents include, for example:1,1′-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl dimethyl),ferrocenyl thiol, and other ferrocene derivatives containing covalentlyattached cryptands. These materials are described, for example, Hammond,et al., J. Chem. Soc. Perkin. Trans. I 707 (1983); Medina, et al., J.Chem. Soc. Chem. Commun. 290 (1991); Shu and Wrighton, J. Phys. Chem.92, 5221 (1988). Included are examples such as the above ferrocenylferrazetine and ferrocenyl cryptand, in which an ordinarily chemicallyinsensitive redox center (ferrocene) is covalently linked to a chemicalrecognition site in such a way as to make the redox center chemicallysensitive. Also suitable are molecules or polymers in which the sensorand reference functionalities are covalently linked such as1-hydro-1′-(6-(pyrrol-1-yl)hexyl)-4,4′-bipyridiniumbis(hexafluorophosphate), as described by Shu and Wrighton, J. Phys.Chem. 92, 5221 (1988).

In some cases, more than one analyte-sensitive redox-active moiety canbe used. For example, there can be one redox-active moiety that issensitive to hydrogen ion, and another that is sensitive to a secondanalyte such as CO, oxygen, ammonia, or an alkali metal. This approachallows for the simultaneous measurement of several analytes. In somecases redox-active agents sensitive to 3 or more analytes can be used.Where there are redox agents sensitive to multiple analytes, in manycases, it is also desired to have one or more analyte-insensitiveredox-active moieties bound to the semiconductor surface as well toprovide a reference, to improve accuracy, and to minimize or avoidcalibration.

Where more than one redox-active moiety is used it can be important toensure there is not significant interference between the peaks. This canbe desirable especially when the multiple redox-active moieties are onthe same electrically addressable zone on the semiconductor substrate.This can be achieved, for example, by ensuring that there is sufficientseparation of the oxidation and reduction potentials, or by physicallyseparating the redox moieties.

In some embodiments, the semiconductor substrate has a plurality ofisolated separately electrically addressable zones. In some embodiments,the different zones will comprise different redox-active moieties. Theuse of separate zones can be beneficial in that the voltammetricmeasurements can be carried out separately, allowing for the use ofmultiple redox-active agents with that have similar reduction and/oroxidation potentials.

The separately electrically addressable zones can be made byconventional semiconductor processing methods, for example masking tocreate structures in specific areas on the surface, for example specificareas on the surface having certain levels of doping. Conventionalsemiconductor processing can also be used to incorporate conductiveinterconnects allowing the zones to be separately addressable. Maskingcan also be used during the attachment of the redox-active moieties tothe semiconductor surface to attach specific redox active moieties todifferent regions of the surface.

The semiconductor substrate with separately addressable zones caneffectively create an electrochemical sensor array. Another aspect ofthe invention provides a semiconductor electrochemical sensor arraywherein a plurality of zones, each zone comprising a redox activemoiety. The array can have multiple zones with analyte-sensitiveredox-active moieties, and one or more zones with analyte-insensitiveredox active moieties. A zone can have a single redox-active moiety, ormultiple redox active moieties. The array can be constructed, forexample, to measure both pH and O₂, wherein one zone comprises aredox-active moiety sensitive to hydrogen ion, another zone has a redoxactive moiety sensitive to O₂, and a third zone with a redox-activemoiety that is insensitive to both hydrogen ion and O₂.

In some embodiments, an array of separately addressable zones or anarray of electrodes is used where there are a plurality of zones orelectrodes that are each constructed to measure the same analyte andused in a redundant matter, wherein another zone capable of measuringthe same analyte is used either simultaneously or in place of the otherzone or electrode. In some cases, more than one zone or electrode, forexample in an array, is used simultaneously to improve the quality ofthe measurement. In some cases, more than one zone or electrode, forexample in an array, is used sequentially, wherein if one zone orelectrode shows degraded performance, the measurement of that analyte isperformed on another zone or electrode constructed to have similarcharacteristics. The sequential use of similar zones or electrodes canprovide reliability of measurement over time. While the electrodes ofthe present invention can be prepared to be robust and to resistfouling, in some circumstances, degradation of the measurement over timemay occur. In some embodiments, the maximum current (I_(max)) can bemonitored over time. A system can be configured, for example, such thatwhen the maximum current drops below a certain level, a switch is madeto a redundant zone or electrode for further measurement of thatanalyte. There can be multiple redundant elements, e.g., 1, 2, 3, 4, 5,10, 20 or more redundant elements.

The semiconductor substrate of the invention can also comprisecircuitry. The circuitry can be used, for example for controlling thecurrent and potential provide to the redox-active moiety. The circuitrycan also be used for analyzing signals or for processing data related tothe voltammetric measurement. The circuitry can also have otherfunctionality, such as the ability to measure other parameters such astemperature, the ability to store date, or the ability to send data andreceive instructions from a remote location. The circuitry can contain,for example, an amplifier such as an operational amplifier. Thecircuitry can contain, for example, an analog to digital converter(ADC). In some cases having the amplifying and ADC functionsincorporated into the semiconductor substrate can provider higherquality and reliability of the transmission of the signal from thesensor. The use of circuitry on the semiconductor substrate can beparticularly useful when an array of zones is utilized. The circuitrycan assist in managing the passage of current in and out of thesemiconductor substrate. In some cases, the circuits can allow forschemes to simultaneously or sequentially address the zones, forexample, by mutiplexing (MUX).

Solid State Electrode Sensors

Another aspect of the invention provides a solid state sensor having asolid state working electrode formed of a solid state (e.g.,semiconductor) substrate. The solid state electrode sensor comprisingthe solid state substrate can be used for the measurement of thepresence or absence of one or more analytes, or can be used toaccurately measure the concentration of analyte in a sample.

In some embodiments, a solid state electrode sensor is formed of asemiconductor, such as silicon. The semiconductor electrode sensor canbe used to detect the presence or absence and/or the measure theconcentration of analytes, including hydrogen ion, alkali metals, CO, orO₂. In some embodiments, the semiconductor electrode sensor is used tomeasure the concentration of hydrogen ion, or pH.

The semiconductor electrode sensor comprises a semiconductor substrateas described above comprising a redox-active moiety that is sensitive tothe presence of an analyte. The semiconductor electrode sensor may alsocomprise a redox active moiety that is insensitive to the presence of ananalyte. The semiconductor electrode sensor can comprise more than onesemiconductor substrate. For example, the sensor may comprise onesemiconductor substrate that has an analyte sensitive redox activemoiety and another semiconductor substrate having an analyte insensitiveredox active moiety.

The semiconductor electrode sensor is configured to be incorporated intoa system that will supply voltage, and can drive current to the sensorto perform voltammetry. Thus, the semiconductor substrate or substrateswithin the sensor may be electrically connected in a manner which willallow for connection to a device for supplying and measuring current andvoltage.

The semiconductor electrode sensor is generally the working electrode inan electrochemical system that will also comprise a counter electrode,and in some embodiments, a reference electrode.

The sensor may be put into contact with a sample having the analyte tobe detected. The sample is generally a liquid sample. In some cases thesample can be a gel, suspension, molten, or semi-solid medium. Thesample can be, for example, a foodstuff. The sample can be any type ofliquid including hydrocarbons, oils, fluorocarbons, silicones, andaqueous solutions. Where the analyte is hydrogen ion, an aqueous mediumis generally used, but in some case a polar protic medium or polaraprotic medium can be used. The sensor is useful for measuring pH inaqueous solutions.

In some embodiments, the sensor of the invention can accurately measureanalyte concentrations where the analyte is present in a concentrationrange from 10⁻¹ M to 10⁻¹⁴ M. In some embodiments, the sensor of theinvention can accurately measure analyte concentrations where theanalyte is present in a concentration range from 10⁻³ M to 10⁻¹⁰ M. Insome embodiments, the sensor of the invention can measure theconcentration to an accuracy of plus or minus 100%, 50%, 30%, 20%, 10%,5%, 2% or 1%. In some embodiments, the sensor of the invention canmeasure the concentration within a range of 10⁻³ M to 10⁻¹⁰ M to anaccuracy of plus or minus 100%, 50%, 30%, 20%, 10%, 5%, 2% or 1%.

In some embodiments, the analyte is hydrogen ion, and the sensor of theinvention can accurately measure the pH in a range from pH 1 to pH 14.In some embodiments, the analyte is hydrogen ion, and the sensor of theinvention can accurately measure the pH in a range from pH 3 to pH 10.In some embodiments, the sensor of the invention can accurately measurepH to an accuracy of plus or minus 0.5, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03,0.02, or 0.01 pH units. In some embodiments, the sensor of the inventioncan accurately measure pH in a range from pH 3 to pH 10 to an accuracyof plus or minus 0.5, 0.3, 0.2, 0.1, 0.07, 0.05, 0.03, 0.02, or 0.01 pHunits.

The semiconductor electrode sensors of the invention can accuratelymeasure analyte concentration in a wide variety of sample types. Thesensors can be made to be robust, and resistant to fouling, andtherefore reliable for long-term measurements.

Another aspect of the invention is a sensor which does not requireroutine calibration (or re-calibration), or in some cases anycalibration at all. Conventional potentiometric sensors rely on a glassmembrane to sense, for example, hydrogen ion. These types of sensorsgenerally need to be calibrated on a regular basis, usually by placingthe sensor into standards of known analyte concentration. These types ofsensors generally need calibration when going from one solution toanother solution, and will also need calibration with time, even if keptwithin the same solution and upon standing outside of a solution. Thesituation is made worse if there is a change in the composition of themedium over the time that the sensor is monitoring the medium, forexample, when monitoring a chemical reaction, biochemical reaction, orfermentation. In these cases, potentiometric sensors may drift and needcalibration due to the accumulation of some species in the reaction, ordue to fouling of the sensor by species present.

In some embodiments, the sensors of the present invention do not need tobe calibrated under any of these situations. In some embodiments, thesensors of the invention do not need to be calibrated over time insolution. In some embodiments, the sensors of the invention do not needto be calibrated after an hour, 10 hours, 1 day, 2 days, 5 days, a week,two weeks, a month, 6 months, 1 year, 2 years or longer while in asolution or in storage. In some embodiments the sensors or the presentinvention are accurate at measuring analyte concentration to 50%, 40%,20%, 10%, 5%, 2%, 1%, 0.5%, 0.2% or 0.1% after the times above. In someembodiments where the sensors measure pH, the sensors are accurate to 1,0, 8, 0.5, 0.3, 0.2, 0.1, 0.08, 0.05, 0.03, 0.02, or 0.01 pH units afterthe times above. In some embodiments where the sensors measure pH, thesensors are accurate to within 0.1 pH units after one week in solutionor in storage.

In some embodiments, the sensor is capable of measuring analyteconcentration without any calibration with an external standard. In someembodiments, the sensor remains sensitive to the analyte withoutcalibration after a first use by an end user.

In some embodiments the analyte is hydrogen ion and the sensor remainssensitive to hydrogen ion after exposure to a cell culture medium for atleast 1, 3, 6, 9, 12, 18, or 24 hours or 2, 3, 4, 6, 8, 12, 24, 48, 60,90, or more days. In some embodiments the analyte is hydrogen ion andthe sensor remains sensitive to hydrogen ion after exposure to a cellculture medium for at least 3 days. In some embodiments the analyte ishydrogen ion and the sensor remains sensitive to hydrogen ion afterexposure to a cell culture medium for at least 6 days. In someembodiments, the sensor is capable of measuring pH with an accuracy of0.2 units after exposure to the cell culture medium.

In some embodiments, the analyte is hydrogen ion and the sensor iscapable of measuring pH with an accuracy of 0.2 units after autoclavetreatment at 121° C. for 10, 20, 40, 80, 100, 200, 400, or 800 minutes.In some embodiments, the analyte is hydrogen ion and the sensor iscapable of measuring pH with an accuracy of 0.2 units after autoclavetreatment at 121° C. for 40 minutes. In some embodiments, the analyte ishydrogen ion and the sensor is capable of measuring pH with an accuracyof 0.2 units after autoclave treatment at 121° C. for 400 minutes.

A subject sensor that does not require calibration over long periods oftime in a medium that can change characteristics is useful, for example,as an implantable sensor. The implantable sensor can be placed under theskin or within the body in contact with a bodily fluid such as blood,saliva, breast milk, amniotic fluid, lymph, sweat, tears, or urine. Thesensor can measure the concentration of analytes such as hydrogen ion,sodium, potassium, calcium, or oxygen.

The implantable sensor has an electrode configured to be in contact witha bodily fluid, said electrode comprising a semiconductor surface thathas immobilized thereon a redox active moiety, wherein the redox activemoiety has an oxidation potential and/or reduction potential that issensitive to concentration of said ion. The implantable sensor can beincluded in an implantable medical device such as described in U.S. Pat.No. 6,738,670. For example, the implantable medical device in which thesensor resides could include pacemakers, defibrillators, drug deliverypumps, diagnostic recorders, cochlear implants, and the like. Theimplantable medical device is typically programmed with a therapy andthen implanted in the body typically in a subcutaneous pocket at a siteselected after considering clinician and patient preferences. In someembodiments the implanted device is in a form which can be swallowed,allowing the measurement of the properties of the regions encountered asit passes through the body such as the digestive tract including thestomach, the upper and lower intestines, and the colon. The informationobtained by the sensor in the implanted device can either be accessed inreal time, for example, by wireless communication, or can be retrievedfrom the device after passage through the body. A wide variety ofprogrammers, also known as downlink transmitters, can be used totransmit data to and receive data from the implantable medical device.Examples of downlink transmitters include devices such as physicianprogrammers, patient programmers, programming wands, telemetry accessunits, and the like. The clinician, for example, can periodically use aphysician programmer to communicate with the implantable medical deviceto manage the patient's therapy and collect implantable medical devicedata. The semiconductor electrode sensor can be incorporated into orattached to the implantable medical device and can provide data onanalyte concentration within the region of the body into which it isimplanted. The patient can use the patient programmer to communicatewith the implanted device to make therapy adjustments that have beenprogrammed by the clinician. Both the physician programmer and patientprogrammer can have an antenna locator that indicates when a telemetryhead is aligned closely enough with the implanted device for adequatetelemetry.

Another aspect of the invention is a method for measuring concentrationin a bodily fluid within a body, the method comprising placing asemiconductor electrode sensor comprising a redox active moiety incontact with the bodily fluid, and operating the sensor to yield a valueof the concentration of the analyte present in said bodily fluid.

Systems for Measuring the Concentration and/or Presence or Absence of anAnalyte

Another aspect of the invention provides a system for measuring analyteconcentration. In some embodiments, the system comprises a workingelectrode having a solid state (e.g., semiconductor) surface that hasimmobilized thereon a redox active moiety, wherein the redox activemoiety has an oxidation potential and/or reduction potential that issensitive to the presence of an analyte; a counter electrode andoptionally a reference electrode. The system further comprises a sourcefor supplying a plurality of potentials to the working electrode, and adevice for measuring current through the working electrode at theplurality of potentials. The working electrode referred to herein cancomprise the solid state electrochemical sensor described above. It isdesirable in some embodiments that the solid state surface also hasimmobilized thereon a second redox active moiety having an oxidationpotential and/or reduction potential that is insensitive to the presenceof said analyte. The redox active moiety that is insensitive to thepresence of the analyte can be on the same solid state surface, or canbe on another solid state surface in electrical communication with thesystem and in contact with the sample. In some embodiments, the redoxactive moiety that is sensitive to the presence of the analyte is onfirst solid state (e.g., semiconductor) working electrode, and the redoxactive moiety that is insensitive to the presence of the analyte is on asecond solid state working electrode that is electrically isolated (orelectrically insulated) from the first working electrode. The system isconfigured such that the working electrode(s), the counter electrode,and optionally the reference electrode are in contact with the sample.In many embodiments, the sample is a liquid sample, and the electrodesare each in contact with the liquid. In some cases, the sample will notbe a liquid, but may be a solid, generally comprising a solidelectrolyte, a semi-solid (e.g., solid-liquid mixture), or a gas, or asample having a viscosity characteristic of a gas or liquid. In someembodiments, the first solid state working electrode is separately andindependently addressable from the second solid state working electrode,enabling a reading from the first working electrode independently fromthe second working electrode.

In some embodiments, the system will have two or more workingelectrodes. For example, in some embodiments, the system will have oneworking electrode comprising a semiconductor surface that hasimmobilized thereon a redox active moiety whose oxidation potentialand/or reduction potential is sensitive to the presence of said analyte,and a second working electrode comprising redox active moiety whoseoxidation potential and/or reduction potential is insensitive to thepresence of said analyte. An example of a system with two workingelectrodes is a system having two semiconductor wafers or chips, one ofwhich has a redox active moiety which is sensitive to pH, such asanthracene, and another redox active moiety which is insensitive to pH,such as a ferrocene. In some cases the semiconductor wafer or chip onwhich each redox active species is immobilized may be a different typeof semiconductor wafer or chip. For instance, the semiconductor wafer orchip to which the pH sensitive moiety is bound may have one dopinglevel, and the semiconductor wafer or chip on which the pH insensitivemoiety is bound may have a different doping level. This type ofconstruction can be beneficial because, in some cases, one type of redoxactive species will perform better in terms of amplitude, sensitivity orstability with one type of doping, while another redox active specieswill perform better on a semiconductor wafer or chip with a differenttype of doping. In some embodiments, the pH sensitive moiety, e.g.,anthracene, is bound to a semiconductor wafer that has a low level ofdoping, and the pH insensitive moiety, e.g., ferrocene is bound to asilicon wafer that has a higher level of doping. In some embodiments thesemiconductor wafer onto which the pH sensitive moiety, e.g., anthraceneis bound has a resistivity between 1 Ω-cm an 1000 Ω-cm, or between 10Ω-cm and 90 Ω-cm, or between 10 Ω-cm and 40 Ω-cm, while semiconductorwafer onto which the pH insensitive moiety, e.g., ferrocene, is boundhas a resistivity between 1 milliohm-cm and 1000 Ω-cm, or 1 Ω-cm and 90Ω-cm, or 10 Ω-cm and 40 Ω-cm. In some embodiments, an N-typesemiconductor (e.g., silicon wafer) is used for a working electrodehaving an H+ insensitive moiety (e.g., ferrocene). In some situations,an undoped or lightly doped p-type semiconductor (e.g., silicon wafer)is used for a working electrode having an H+ sensitive moiety (e.g.,anthracene). In other embodiments, a P-type semiconductor (e.g., siliconwafer) may be used for both the pH sensitive and the pH insensitivemoieties.

In some embodiments, the system will have 3 or more working electrodes.For example, in some embodiments, the system will have one workingelectrode comprising a semiconductor surface that has immobilizedthereon a redox active moiety that is sensitive to the presence of afirst analyte, a second working electrode comprising a semiconductorsurface that has immobilized thereon a redox active moiety that issensitive to the presence of a second analyte, and a third workingelectrode comprising a semiconductor surface that has immobilizedthereon a redox active moiety that is insensitive to the presence ofeither the first analyte nor the second analyte. The system can alsohave more than 3 working electrodes, for example having 4, 5, 6, 7, 8,9, 10, 12, 20, 50 or more working electrodes, each having redox activemoieties sensitive to and analyte. These systems can also have one ormore than one semiconductor working electrode having a redox speciesthat is insensitive to the analytes, for example to provide a reference.In some cases more than one redox species that is insensitive to theanalyte can be used.

In some embodiments the system of the present invention comprises aprobe that comprises the 2 or more electrodes. The probe can physicallyhold the electrodes such that the electrodes can be brought into contactwith the sample. The probe allows the working electrodes to be heldclose to the reference and/or counter electrode. FIG. 21 shows anexemplary embodiment of a probe of a system of the present inventionhaving 4 electrodes: a first working electrode (WE1), a second workingelectrode (WE2), a reference electrode (RE), and a counter electrode(CE). WE1 and WE2 can each be formed of a solid state material, such asa semiconductor. FIG. 21( a) shows a shaded drawing of the probe. FIG.21( b) shows a line drawing of the probe. As shown in FIG. 21, in somecases it can be beneficial in the present invention for the referenceelectrode and the counter electrode to have a ring configuration. Inother embodiments, only one of the reference electrode or counterelectrode will have a ring configuration. A ring electrode can in somecases provide signal stability. While this embodiment shows oneconfiguration, there are other ring electrode configurations that can beused with the present invention.

In some embodiments it is useful to use undoped or lightly dopedsemiconductor (e.g., silicon) substrates for a moiety such asanthracene. While not being bound by theory, the band gap of thesemiconductor, e.g., silicon can be influenced by the level of doping ofthe semiconductor, and it is believed that in some cases, the use of asemiconductor with the appropriate level of doping can be useful totailor the appropriate redox active moiety with the appropriate bandgap. Thus in some cases it is desirable to use lightly dopedsemiconductor, e.g., silicon with a moiety such as anthracene. Thus insome embodiments it is useful to use two semiconductor workingelectrodes: one optimized for ferrocene moieties and the other optimizedfor the anthracene moieties. When two working electrodes are used, insome embodiments, two sequential electrochemical measurements (e.g.,with square wave voltammetry) may be carried out using the same counterand reference electrode. For instance, the first measurement can beconducted using reference, counter and working electrode 1 (e.g.,anthracene derivatized) between −1.2 to −0.5 V, followed by the secondmeasurement which may be conducted using reference, counter and workingelectrode 2 (e.g., ferrocene derivatized) between 0 to 0.5 V. The peakspotential detected in the first and second measurements can then bestored and processed to get a pH reading. This type of system and methodcan be accomplished through the use of a bipotentiostat or a two-channelmultiplexer. A similar approach can be applied to multiple workingelectrodes with a mutipotentiostat or a multi-channel multiplexer.

The system is configured to carry out voltammetric measurements on thesample. Some embodiments provide a method which includes the measurementof pH with a voltammetric pH sensing system comprising the semiconductorelectrode sensor described above, a potentiostat for providing voltageto the electrodes, and a meter for detecting the current as a functionof voltage.

The counter electrode typically is needed to complete theelectrochemical circuit to make the measurements described herein. Thecounter electrode is generally made of a material which iselectrochemically inert to the medium so that current overloading doesnot occur during the course of measurement. Suitable materials in manyapplications include platinum, silver, gold, stainless steel, andcarbon.

A reference electrode is optional and is used as a third electrode insome embodiments of the invention. In the case of a three-electrodesystem, the counter electrode generally completes the circuit, allowingcurrent to flow through the cell, while the reference electrodemaintains a constant interfacial potential difference regardless of thecurrent. In the case where the system comprises an analyte sensitiveredox active moiety and an analyte insensitive redox active moiety, theanalyte insensitive redox active moiety can act as a reference, allowingthe potential difference to be used to determine analyte concentration.Even where the system comprises an analyte insensitive moiety, in someembodiments, a reference electrode will still be used. In someembodiments, pseudo-reference electrodes can also be utilized. Referenceelectrodes that can be employed include: Standard hydrogen electrode(SHE), also known as “normal hydrogen electrode” (NHE), saturatedcalomel electrode (SCE), copper-copper(II) sulfate electrode, andsilver/silver chloride (Ag/AgCl) electrode. In some embodiments a silverelectrode or a polyurethane coated silver electrode can act as asilver/silver chloride electrode where sufficient chloride is present ator near the silver electrode. In some cases, e.g., in non-aqueousmedium, a metal electrode such as a platinum or a silver electrode, or apolyurethane coated silver electrode can be used as the referenceelectrode.

To carry out voltammetry, the system generally has a source forsupplying a plurality of potentials. The voltammetry can be, for examplecyclic voltammetry, pulse voltammetry, normal pulse voltammetry, squarewave voltammetry, differential pulse voltammetry, linear voltammetry, orsquare wave voltammetry. The source for supplying a plurality ofpotentials can be a potentiostat, for example, a potentiostat capable ofapplying square waves for square wave voltammetry.

Generally, the analyte concentration is determined by using voltammetryto identify the position of current peaks, which current peaks indicatethe reduction or oxidation potential of a redox active moiety. In someembodiments, the position of the reduction and/or oxidation potential ofthe analyte sensitive redox active moiety is used to determine theconcentration of the analyte. This method can be used, for example,where no analyte insensitive redox active moiety is employed.

Where an analyte insensitive redox active moiety is used, detection isgenerally accomplished by measuring the potential difference, delta E,associated with current peaks for oxidation (or reduction) of theimmobilized redox active moieties, where the magnitude of delta E can berelated to the concentration of analyte, e.g., hydrogen ion (H+) insolution. The analyte insensitive redox active moiety has anelectrochemical response that is insensitive to variations in the mediumand serves as the reference. Current peaks for oxidation or reduction ofthe reference and indicator are determined from a voltammogram using acounter electrode.

In some embodiments, the system further comprises a computation systemthat communicates with the device for measuring current. The computationsystem can have algorithms for calculating reduction or oxidationpotential from the measured current at a plurality of potentials fromthe voltammetry measurements. The computing systems can be part of thesensing system, in some cases allowing the sensing system to beself-contained. The computing system can comprise memory for storing rawand/or processed data from the sensors. The computing system can beconnected to a transmission device that will wirelessly, by wire, fiberor other means, transmit processed data to an external device. Thecomputing system can provide signals and measurements which can betransmitted in some cases in real time, allowing the system to alert endusers of conditions which may require attention. The transmitted signalsand measurements can, for example, provide the information required foradjusting a manufacturing process such as a chemical or biochemicalprocess.

In some embodiments the system is made up of a housing that holds thesemiconductor electrode sensor that is electrically connected to a unitcomprising the source for supplying a plurality of potentials and thecurrent measuring device. In some embodiments the unit also comprisesthe computing system described above for at least partially analyzingthe data. The unit can be self powered, e.g., with a battery, or canhave a connection to an outside power source. The unit can have adisplay and input buttons to allow the user to control the measurementand to read the output from the sensor. The unit can have transmissioncapability for sending out data, and for receiving instructions or to betested by an external device.

FIG. 1 shows drawings of an embodiment of the connection of asemiconductor sensor electrode into a sensor housing for use inmeasuring analytes in a fluid such as the fluid in a biochemicalreactor. FIG. 1( a) shows a blow up drawing of an assembly that holdsthe semiconductor electrode sensor and provides electrical connectionsto the semiconductor electrode sensor for voltammetry measurements. InFIG. 1( a) the semiconductor electrode sensor (I), is held in place bythe end cap (II), in contact with the metallized ceramic disk (III). Theceramic disk can be metallized in specific areas on both the front andthe back of the disk, with vias connecting the specific metallizedareas. On one side of the ceramic disk, the semiconductor workingelectrodes as well as the counter and optionally reference electrodescan be present. For example, electrodes such as the semiconductorworking electrodes can each being mounted to a specific metallized area.The disk is then mounted into the housing such that the side of the diskhaving the electrodes is exposed to the medium into which the probe isimmersed, and the other side of the disk is away from the medium,allowing for electrical connection to the metallized areas on to thedisk such that voltage can be applied and current can flow to and fromthe electrodes. The sealing gasket (IV) provides sealing from the fluidin which the sensor is immersed while allowing electrical contact withthe pin contacts (V) on the shaft of the housing (VI). FIG. 1( b) showsthe housing assembled for insertion into the fluid to be measured. Pipefittings are used to seal the wires that provide electrical connectionto the semiconductor electrode for voltammetric measurements.

FIG. 2 shows a drawing of an embodiment of the unit comprising thesource for supplying a plurality of potentials and the current measuringdevice. FIG. 2( a) shows a top view and FIG. 2( b) shows a back sideview. The unit has an electrical input/output connector for connectingto the electrodes for carrying out voltammetry. The unit has aconnection for AC power. The unit has a universal serial bus interface(USB I/F) and an RS-232 Serial port for transmitting data, for receivinginstructions, and for testing and debugging by an external device. Theunit also comprises a liquid crystal display (LCD) and has userinterface buttons to allow the user to control the measurements and toread the output from the sensor.

FIG. 3 shows another exemplary embodiment of a system, in accordancewith an embodiment of the invention. FIG. 3 shows a probe that containstwo semiconductor (silicon) working electrodes, a reference electrode,and a counter electrode. The electrodes can be electrically connectedthrough the probe to the source for supplying a plurality of potentialsto the working electrode, the counter electrode, and optionally thereference electrode, and a device for measuring current through theworking electrode at the plurality of potentials. In this embodiment,the two working electrodes, the reference electrodes and the counterelectrode are contained on the end of the probe on a disk which allowsthe electrodes to be in contact with the medium comprising theanalyte(s) to be measured. The areas of the various electrodes can bevaried to improve the performance of the system. While this embodimentshows two working electrodes, in some embodiments, there may be oneworking electrode, and in other embodiments, there are 3, 4, 5, 10, 20,or more working electrodes. In some embodiments one working electrodecan comprise a semiconductor surface with a redox active moiety that issensitive to pH, such as anthracene, and the other working electrode cancomprise a redox active moiety that is insensitive to pH, such asferrocene. In some embodiments, the semiconductor surface on which thepH sensitive moiety, such as anthracene, is bound comprises a siliconwafer that is lightly doped, and the silicon surface on which the pHinsensitive moiety, such as ferrocene, is bound comprises a siliconwafer that is more heavily doped.

The semiconductor working electrodes can be bonded to conductive regionson the disk. Conductive vias through the disk allow electricalconnection to electrode pins which are contained within the housing. Theelectrode pins are in turn, electrically connected, for example towires, which can run through the probe housing, and then out of thehousing to electrically connect the electrodes to the source forapplying potentials to the electrode, and to the device for measuringthe current that passes through the electrodes. The threads, o-ring, andhex body allow for the probe to be mounted into a reactor such as abioreactor or fermentor. The threads allow for the probe to mate with acorresponding threaded hole through the wall of the reactor, the hexbody allows for tightening the probe into the reactor, and the gasketassists in establishing a seal. In some embodiments the unit is mountedinto the reactor without the use of threads, mounting without threadscan provide resistance to some failure modes that are present whenthreads are used.

In some embodiments, the system is configured to be used as an in-linesensor in a process. An in-line sensor can be a sensor that is used inan on-going process. In some embodiments the sensor is in a vessel, inother embodiments the sensor is in a conduit or pipe through which aprocess fluid flows. In some embodiments, the currents measured at aplurality of potentials by voltammetry are used to determine analyteconcentration, and the determined analyte concentration is used tocontrol a process parameter. The systems of the present invention arevaluable in in-line sensing in that they can be made to be robust, toresist fouling, and are able to measure analyte concentration for longperiods of time in media that changes its properties, as in a processsuch as a chemical reaction.

Methods for Forming Solid State Electrochemical Sensors

Another aspect of the invention provides a method for forming a solidstate electrochemical sensor. The method generally comprises having asolid state (e.g., semiconductor) substrate with a surface andimmobilizing a redox active moiety with a reduction and/or oxidationpotential that is sensitive to an analyte onto the solid state surface.

Some embodiments provide a method for forming an analyte-sensitivesemiconductor electrode, the electrode having a semiconductor surface.The method comprises immobilizing a redox-active moiety that issensitive to the presence of an analyte onto the semiconductor surface.

Any suitable method such as those known in the art or disclosed hereincan be used to construct a semiconductor surface as described above thatis useful as part of a subject sensor. The redox groups can beimmobilized onto the surface chemically or physically. The redox groupscan be reacted with the semiconductor surface to attach them to thesemiconductor covalently. Alternatively, the redox active groups can beadsorbed to the semiconductor. The redox active groups can also beimmobilized by attaching the groups to a polymer that is eithercovalently or non-covalently bound to the surface. Covalent binding ofeither the redox group or the polymer to which the redox group is a partto the surface can be beneficial in improving the lifetime and stabilityof the electrode.

Functional groups can be covalently attached to semiconductors, such assilicon or germanium. Silicon can, for example, form covalent bonds withcarbon, and thus is a desirable substrate for functionalizing withcarbon based molecules. The covalent binding to the surface can bethrough a bond between the semiconductor, e.g., silicon, and carbon,oxygen, nitrogen, sulfur, or other atom. In some embodiments the bond tothe surface is between the semiconductor, e.g., silicon, and carbon. Insome embodiments the bond to the surface is between semiconductor, e.g.,silicon, and oxygen.

In some embodiments, the immobilization of the redox active moiety bycovalent binding to the semiconductor surface is accomplished byreaction with a semiconductor hydride, for example, a silicon hydride(Si—H) surface. A semiconductor-hydride, e.g., silicon-hydride surfacecan be obtained, for example by treatment of the semiconductor, e.g.,silicon surface, for example a surface that is in the native oxidestate, with hydrofluoric acid (HF). For example, dilute (1-3%) aqueousHF treatment, or, a 40% aqueous NH₄F treatment can be used to create aSi—H terminated surface. Porous silicon, when etched through standardprocedures involving HF, can also be used as a Si—H surface. FIG. 4shows a schematic illustrating the conversion of a native oxide surfaceto a Si—H surface on a silicon wafer through the treatment of the waferwith a 2.5% aqueous solution of HF. A Si—H surface can also be formed byother processes, for example by decomposition of silanes as described inU.S. Pat. No. 6,444,326. A Si—H surface can also be formed throughreacting surface silanol moieties with reagents such astrihydroxyhydridosilane via sol-gel type methods (see e.g., U.S. Pat.No. 5,017,540, and U.S. Pat. No. 5,326,738) or through thy-etchprocesses with plasmas of sulfur hexafluoride or Freon 23. A Germaniumhydride (Ge—H) surface can undergo the same types of the reactions ofSi—H to create covalently bonded redox active reagents. Suitablereactions to covalently bond to the Si or Ge surface are described, forexample, in J. M. Buriak, Chem. Review 2002, 102 (5), 1271. The hydridesof other semiconductor substrates can also be prepared and used tocovalently bond redox active moieties. Suitable semiconductor hydridesinclude for example, hydrides of silicon germanium (M. S. Carroll, etal., J. Electrochem Soc. 2000, 147 (12), 4652), gallium arsenide (P. E.Gee, et al., J. Vacuum Sci. Tech. A: Vacuum Surf. Film 1992, 10 (4),892), gallium nitride, diamond film (S. Yamashita, et al., U.S. Pat. No.5,786,604 (1998)), and indium phosphide (Y. Sun, et al., J. Appl. Phys.2005, 97, 124902).

The semiconductor-hydride surface, e.g., Si—H surface, can be reactedwith a variety of functional groups to create covalent bonds and therebyattach a redox active moiety to the semiconductor surface. The Si—Hsurface, Ge—H surface or other Semiconductor-H surface can participatein hydrosilylation reactions, involving the addition of, for example,the Si—H across an unsaturated site to form a Si—C, Si—O, or Si—N bondto the surface. Functional groups which can be used in these reactionsincluding hydrosilylation include alkenes, alkynes, imines, carbonylsand oximes. These reactions, including hydrosilylation can be carriedout thermally, photochemically, with a metal catalyst or with a radicalinitiator (see Buriak, Chem. Commun., 1999, 1051-1060). The Si—H surfaceor other semiconductor-H surface can also be reacted with alkyl or arylcarbanions through, for example, Grignard, or lithium reagents. In someembodiments, a Si—H surface or other semiconductor-H surface can reactwith azido, diazo, and diazonium groups. Suitable diazonium reactionsare described, for example, in Stewart et al., J. Am. Chem. Soc. 126,2004, 370-378.

FIG. 5 illustrates the reaction of a surface Si—H with an aldehydefunctionality attached to a ferrocene redox active moiety to create acovalently bound ferrocene through a Si—O bond. FIG. 5 also illustratesthe reaction of a surface Si—H with a vinyl functional group attached toa ferrocene redox active moiety to create a covalently bound ferrocenethrough a Si—C bond. FIG. 6 illustrates the reaction of a surface Si—Hwith an aldehyde functionality attached to an anthracene redox activemoiety to create a covalently bound anthracene through a Si—O bond. FIG.6 also illustrates the reaction of a surface Si—H with a vinylfunctional group attached to an anthracene redox active moiety to createa covalently bound anthracene through a Si—C bond. FIG. 7 illustratesthe reaction of both a ferrocene redox active moiety and an anthraceneredox active moiety through vinyl functionality to produce a siliconsurface with a covalently attached redox group that is sensitive tohydrogen ion (anthracene) and a covalently attached redox group that isinsensitive to hydrogen ion (ferrocene). In some embodiments, a carbonylgroup such as an aldehyde group is substituted for the vinylfunctionality for providing attachment to the surface.

The redox active moieties can alternatively be attached covalently tothe surface by direct reactions with a semiconductor, e.g., siliconsurface from which all functionality has been removed, typically by hightemperature and vacuum. The pure silicon surface, for example, can reactdirectly, for example with alkenes and alkynes to form Si—C covalentattachment. (see Bateman, et. al., Angew. Chem. Int. Ed. 1998, 37(19),2683-2685). Diazonium species can also be used to functionalize thesurface either thermally or electrochemically. In some embodiments,ultrahigh vacuum techniques can be used to prepare the functionalizedsurfaces of the invention, for example by [2+2] Reactions of alkynes andalkenes or Diels-Alder ([4+2]) reactions of dienes with reconstructed Sisurfaces.

Capping of semiconductor oxide surfaces such as silica and glasssurfaces with alkyl-, alkoxy- and chloro-silanes may also be used tofunctionalize the semiconductor surface.

The semiconductor surface can contain oxide functionality includinghydroxy functionality (native oxide). In some embodiments, thesemiconductor electrodes of the invention are modified by covalentattachment to this oxide functionality. For example, the hydroxy groupsof silicon or other semiconductor elements can be coupled to surfacebound groups using the many reactions known in organic chemistry forcarbon bound hydroxy groups, including for example, the formation ofesters and ethers. One derivatization method involves the use of acarbodiimide for coupling to the surface. Exemplary carbodiimidesinclude, for example, dicyclohexylcarbodiimide (DCC), or(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC).

The redox active moieties can also be attached covalently to the surfaceby reactions with the native oxide, —O or —OH on the semiconductorsurface. Many methods are known for carrying such reactions to formcovalent bonds through various functional groups (see Maoz et al., J.Colloid. Interface Sci., 1984, 100, 465-496). In some embodiments anindirect approach can be employed in which an alkoxysilane comprisingother reactive functionality is reacted with the —O or —OH groups on thesemiconductor surface to covalently attach the alkoxysilane to thesemiconductor surface. The other reactive functionality on thealkoxysilane can then be used to covalently attach a redox active moietyto the surface. In these embodiments, the alkoxysilane can become alinker or portion of a linker. The other reactive functionality can beany reactive functionality that can be used to attach a redox activemoiety. The functionality can be, for example, an olefin, an acetylene,an amine, a mercaptan, or an epoxy group. The reaction used to couplethe alkoxysilane to the redox active moiety can be, for example a,Diels-Alder reaction, Michael addition, click chemistry, or epoxychemistry. Semiconducting polymers comprising redox active moieties canbe polymerized onto a surface, graft polymerized onto a surface,photo-polymerized, or pre-formed and cast onto a surface.

In some embodiments, the reactions described above for covalentlyattaching functional groups to the semiconductor surface are used toattach a linker group or portion of a linker group having a chemicalfunctionality that can be used to covalently bind the redox activemoiety to the surface in a subsequent step.

Reactions that can be used to covalently attach a redox active moiety tothe semiconductor surface include hydrosilylation, free radicalreactions, carbodiimide coupling, Diels-Alder reactions, Michaeladdition, epoxy reactions, or click chemistry (see, e.g., Evans et al.Australian Journal of Chemistry 60 (6): 384-395 (2007).

Organic semiconductor substrates of the invention can in someembodiments be modified to attach redox active species after theformation of the organic semiconductor. For example, an organicsemiconductor can be cast into a film. In some embodiments the organicsemiconductive polymers, for example, polyaniline, polypyrrole, andpolythiophene are prepared by chemical or electrochemical oxidation. Thesemiconductive polymer can be subsequently modified using, for example,the chemistry described for the attachment of redox active agents to theinorganic semiconducting substrates. In some embodiments, thesemiconductive polymers can comprise functional groups that can be usedto attach redox active species to the semiconductive polymer. Forexample, the sulfonic acid groups in poly(anilinesulfonic acid) can beused to immobilize aminoferrocene, 1,1′-diaminoferrocene, andaminoanthracene through sulfamide formation. In some embodiments, apyrrole, thiophene or aniline monomer can have a functional group suchas an N-hydroxy succinimide ester, a carboxylic acid, or a reactivevinyl or allyl group. The monomers can then be polymerized, for exampleelectropolymerized, and the redox active moiety can be attached to thefunctional group on the polymerized monomer.

In some embodiments, the redox active moiety can be incorporated intothe semiconductive polymer itself, for example as part of the backboneof the polymer. For example, redox active groups within poly(ferrocenylvinylene phenylene vinylene), poly(fluorine), and poly(carbazole) thatcomprise redox active moieties in their main chains, which can be usedfor analyte, e.g., pH sensing.

In some cases the surface of the semiconductor is modified by coatingthe surface with conductive compounds such as metals or metal oxides. Insome embodiments, the semiconductor is coated with gold, silver,palladium, copper, platinum or other metals. The metals can be coatedfrom solution, for example by electrodeposition, or can be coated ontothe surface with vacuum techniques such as plasma deposition, or metalvaporization. The semiconductor surface can be coated with conductive orsemiconductive metal oxide compounds such as indium-tin oxide. Whenthese materials are coated onto the semiconductor electrode surface, theredox-active agents are attached to the semiconductor electrode byattachment to the layer on the semiconductor electrode.

Where a linker group is used, the linker can be small, for example oneto 3 atoms, or can be longer, e.g., 20 to 100 atoms. The linker can alsobe any size between the small or longer linker. In some embodiments, thelinker is relatively short allowing for the redox active moiety to beclose to the surface, which can be beneficial for electron transfer. Insome embodiments the linker is provided such that the redox active agentis held 1, 2, 3, 4, 5, 6, or 7 atoms from the surface of thesemiconductor. Where a short linker is used, the redox-active moiety isheld close to the surface. Where a longer group is used, the redoxactive moiety may be able to move away from the surface, for examplefurther out into the solution. Linker groups can comprise hydrophilic,hydrophobic groups, or mixtures thereof. Linker groups can comprise, forexample, hydrocarbons, alkenes, alkynes, esters, ethers, amides, amines,carbonyls, thiols, olefins, silicones, or other organic, inorganic ororganometallic groups. The linker groups can be formed by polymerizationor oligomerization reactions such as free radical or anionicpolymerization. The linker group can comprise, for example, ethyleneoxide, propylene oxide, or acrylamide repeat units. Linkers can havering structures including aromatic rings. The variation in the linkerstructure can be used to vary the mobility of the redox-active moiety inthe solution. If the linker is too long and densely packed the redoxactive moiety can be far enough away from the surface such that theelectron transfer to the electrode surface may be compromised. In thesecases, having a linker that has electrical conductivity can be useful.

A redox-active moiety may be highly substituted, and can still act as aredox-active moiety. Thus, for example, the redox-active moietyferrocene includes substituted ferrocenes, ferrocene polymers, andferrocene covalently attached to the surface via linker molecules.

In some embodiments, the redox-active moiety can be incorporated into apolymer, and the polymer comprising the redox active moiety can beimmobilized onto the semiconductor surface. The immobilization of thepolymer can be either chemical or physical. The immobilization of thepolymer can be through covalent bonds, or through adsorption of thepolymer to the semiconductor surface.

The redox active moieties can be incorporated into any type of polymerthat can be immobilized onto the surface of the semiconductor surface.Types of polymers that the redox active moieties can be incorporatedinto include biopolymers such as RNA, DNA or proteins, conductivepolymers, fluoropolymers, polyterpenes, inorganic polymers, phenolicresins polyanhydrides, polyesters, polyolefins, polysiloxanes,polyamides, polyimides, polyethers, polyketones, polysulfones, and vinylpolymers.

The polymer comprising the redox active moieties can, in some cases, beproduced at the semiconductor surface. For example monomers or oligomerscomprising the redox active moieties can be polymerized in the region ofthe surface to product the polymer near the surface. In some cases, thepolymerization can be initiated at the semiconductor surface, resultingin polymer covalently bound to the surface. The polymerization can beinitiated at the surface can be initiated, for example by a free radicalreaction initiated by a diazo group attached to the semiconductorsurface. In other cases, the polymerization can be initiated insolution, for example near the surface, such that the nascent polymer isimmobilized onto the surface as it is formed. Methods for determiningthe appropriate solvent conditions are known. For example byestablishing that the monomer and/or oligomer are soluble, while thepolymer is insoluble, allowing for surface deposition to occur. Thesemiconductor surface can comprise polymerizable functional groups thatare capable of copolymerizing with the monomers or oligomers comprisingthe redox active moieties resulting in covalently binding the redoxactive polymer onto the semiconductor surface.

In some embodiments, a polymer comprising the redox active moieties canbe electropolymerized at the semiconductor surface. For example,monomers comprising the redox active moieties are added to a solution,and current is provided through the semiconductor surface causingelectropolymerization of the monomers. In some embodiments, theelectropolymerization can result in the covalent attachment of theelectropolymerized polymer to the semiconductor surface. In otherembodiments, the electropolymerization can result in polymerization, forexample, at the semiconductor-solution interface, and the polymer thatis formed can deposit onto the semiconductor surface, resultingimmobilization of the polymer by physisorption to the surface. Thepolymers can be polymerized onto a surface, graft polymerized onto asurface, photo-polymerized, or pre-formed and cast onto a surface.

The polymers can be chemically or electrochemically deposited onindividual microelectrodes, polymerized, as respond to a signal in areversible manner, in a way that can be electrochemically detected.Other such materials are described by R. W. Murray in ElectroanalyticalChemistry, vol. 13, edited by A. J. Bard (Marcel Dekker, NY 1984), theteachings of which are specifically incorporated herein.

In some embodiments, the polymer is formed away from the semiconductorsurface, and subsequently immobilized thereto. The polymer can beimmobilized onto the surface by a variety of methods including,adsorption from solution, coating including spin coating and dipcoating, spraying, printing, electropainting, or electrodeposition.

In some embodiments the semiconductor electrode is formed by contactingan H-terminated semiconductor surface with the one or more redox-activemoieties wherein at least one redox active moiety is sensitive to thepresence of an analyte, wherein each redox-active moiety comprises afunctional group that will react with the H-terminated semiconductorsurface to form a covalently bond, thereby forming a derivatizedsemiconductor surface. In some embodiments, the surface comprises atleast two redox active moieties and one of the redox active moieties isinsensitive to the presence of the analyte.

Methods for forming semidonductor electrochemical sensors can be appliedto forming other solid state electrochemical sensors, such as carbonsensors. It will be understood that in the case of other solid statematerials, the doping configuration can be changed to effect desirablesensor properties, such as sensitivities and reliability.

Uses of the Compositions and Devices

Another aspect of the invention provides a method for determining theconcentration of an analyte. In an embodiment, the method comprisesbringing an electrode of a sensor in contact with the analyte, theelectrode comprising a solid state (e.g., semiconductor) substrate withsurface having immobilized thereon an analyte-sensitive redox-activemoiety. The analyte-sensitive redox-active moiety exhibits an oxidationpotential and/or reduction potential that is sensitive to the analyte.Next, a plurality of potentials are applied to the electrode. Thecurrent through the electrode is measured at the plurality of potentialto determine a reduction and/or oxidation potential of theanalyte-sensitive redox-active moiety, thereby determining theconcentration of the analyte.

In some embodiments, the method comprises determining the concentrationof an analyte by (a) placing an electrode in contact with said analyte,said electrode comprising a solid state (e.g., semiconductor) substratewith surface having immobilized thereon an analyte-sensitiveredox-active moiety, said analyte-sensitive redox-active moietyexhibiting an oxidation potential and/or reduction potential that issensitive to the concentration of the analyte; (b) applying a pluralityof potentials to the electrode; and (c) measuring the current throughthe electrode at the plurality of potentials, and determining areduction and/or oxidation potential of the analyte-sensitiveredox-active moiety, thereby determining the concentration of theanalyte.

The method of determining the concentration of the analyte can be usedto measure pH by utilizing redox active moieties that are sensitive tohydrogen ion as described above.

The measurement of current at a plurality of potentials allows forcarrying out voltammetry for determining the oxidation and or reductionpotential of the redox active moiety or moieties immobilized on thesurface. The voltammetry used in the method can be, for example cyclicvoltammetry, pulse voltammetry, normal pulse voltammetry, square wavevoltammetry, differential pulse voltammetry linear voltammetry, orsquare wave voltammetry. The source for supplying a plurality ofpotentials can be a potentiostat, for example, a potentiostat capable ofapplying square waves for square wave voltammetry.

The frequency of the measurement can affect the quality of the data. Insome embodiments, square wave voltammograms are currently at a stepheight of 2 mV, amplitude of 25 mV and a frequency of 10 Hz. In somecases, it is advantageous to increase the frequency. For example,increasing the frequency to 500 Hz can result in a faster scan rate. Wehave observed that in some cases, a higher frequency results in a higherlevel of observed current. In some cases, the peak current can shift,for example to more negative potentials upon increasing the operatingfrequency. In some cases changing the potential at higher frequenciescan result in more noise in the square wave voltammograms. In some casesit is advantageous to shield the electrode from the light because insome cases, light can contribute to the background noise.

In some embodiments, the electrode further comprises ananalyte-insensitive redox-active moiety having a reduction and/oroxidation potential that is substantially insensitive to the analyte,and the method further comprising determining the oxidation and/orreduction potential of the analyte-insensitive redox-active moiety, anddetermining the concentration of the analyte from the difference in theoxidation and/or reduction potentials of the analyte-sensitive andanalyte-insensitive moieties. The redox-active moiety having a reductionand/or oxidation potential that is substantially insensitive to theanalyte can be on the same electrode as the redox-active moietyexhibiting an oxidation potential and/or reduction potential that issensitive to the analyte, or a different electrode (e.g., separate andelectrically isolated electrodes).

Generally, the analyte concentration is determined by using voltammetryto identify the position of current peaks, which current peaks indicatethe reduction or oxidation potential of a redox active moiety. In someembodiments, the position of the reduction and/or oxidation potential ofthe analyte sensitive redox active moiety is used to determine theconcentration of the analyte. For example, the position of the currentpeak with respect to the potential at a reference electrode can be used.This method can be used, for example, where no analyte insensitive redoxactive moiety is employed.

Where an analyte insensitive redox active moiety is used, detection isgenerally accomplished by measuring the potential difference, delta E,associated with current peaks for oxidation (or reduction) of theimmobilized redox active moieties, where the magnitude of delta E can berelated to the concentration of analyte, e.g., hydrogen ion (H+) insolution. That is, in many embodiments, delta E represents the potentialdifference between the reduction and/or oxidation potential between aredox active analyte sensitive moiety and a redox active analyteinsensitive redox active moiety. The analyte insensitive redox activemoiety which has an electrochemical response that is insensitive tovariations in the medium serves as the reference. Current peaks foroxidation or reduction of the reference and indicator can be determinedfrom a voltammograms using a counter electrode, and without the need fora reference electrode.

In some embodiments the measured current through the electrode at theplurality of potentials is used to determine the concentration of theanalyte. The determination of the concentration using the measuredcurrent (e.g., current peaks) can be accomplished by using a computationsystem that communicates with the device for measuring current. Thecomputation system can apply algorithms for calculating reduction oroxidation potential from the measured current at a plurality ofpotentials from the voltammetry measurements. The computing systems canbe part of the sensing system, in some cases allowing the sensing systemto be self contained. The computing system can utilize its memory forstoring raw or processed data from the sensors. The method can furthercomprise communication between the computing system and the sensor viatransmission device that will wirelessly or by wire transmit processeddata to an external device.

Carrying out the method typically requires the use of at least one otherelectrode (the counter electrode). The counter electrode is used tocomplete the electrochemical circuit to make the measurements describedherein. The counter electrode is generally made of a material that ischemically inert to the medium so that its potential does not changesignificantly during the course of measurement. Suitable materials inmany applications include platinum, gold, stainless steel, and carbon.In some cases, the counter electrode can be incorporated into the chipthat also comprises the semiconductor sensor electrode.

A reference electrode is optional and is used as an additional electrodein some embodiments of the method of measuring analyte concentration. Inthe case of a three-electrode system, the counter electrode generallycompletes the circuit, allowing current to flow through the cell, whilethe reference electrode maintains a constant interfacial potentialdifference regardless of the current. In the case where the systemcomprises an analyte sensitive redox active moiety and an analyteinsensitive redox active moiety, the analyte insensitive redox activemoiety can act as a reference, allowing the potential difference to beused to determine analyte concentration. Even when an analyteinsensitive moiety is also used, in some embodiments, a referenceelectrode will still be used. In some embodiments, pseudo-referenceelectrodes can also be utilized. Reference electrodes which can beemployed are described above.

In some embodiments, the sample is a liquid sample, and the electrodesare each in contact with the liquid. In some cases, the sample will notbe a liquid, but may be a solid, generally comprising a solidelectrolyte, or a gas.

In some embodiments, the method involves the in-line sensing of aprocess. An in-line sensor can be a sensor that is used in an on-goingprocess. In some embodiments the method comprise the use of a sensor isin a vessel, in other embodiments the sensor is in a conduit or pipethrough which a process fluid flows. In some embodiments, the methodcomprises using currents measured at a plurality of potentials byvoltammetry to determine analyte concentration, and the determinedanalyte concentration is used to control a process parameter. Thesystems of the present invention are valuable in in-line sensing in thatthey can be made to be robust, to resist fouling, and are able tomeasure analyte concentration for long periods of time in media thatchanges its properties, as in a process such as a chemical reaction,biochemical reaction, or fermentation.

In some cases, under remote monitoring the sensor can be programmed toautomatically take readings. The automatic readings can be programmed tooccur on a periodic basis, to occur upon the happening of some event, orto occur when the sensor is prompted. The periodic events can beseparated on the order of seconds to on the order of months. Thehappening of some event could be, for instance at the point when themeasured solution reaches a certain volume level, or at given points inthe steps of a manufacturing process (e.g., at the beginning of or endof a step, or upon the addition of a reagent to a vessel).

Remote monitoring generally includes communication from the remotesensing unit, and/or communication to the remote sensing unit. Thecommunication to and from the remote sensing unit can be done withtransmission lines, and/or wirelessly. Any type of signal including, forexample, digital, analog, wideband, narrowband, or combinations thereof,can be used.

Another aspect of the invention provides voltammetric monitoring of pHwith a semiconductor electrode as part of process control in processes.In an embodiment, a voltammetric pH measurement is made in an industrialprocess stream, and the pH value from that measurement is used to asinput to a decision on the adjustment a process parameter. In anembodiment, the pH value from the voltammetric pH measurement with asemiconductor electrode is used to decide whether or not to add one ormore components to the process, and/or to decide how much of thecomponent to add. In some embodiments, the pH value is used to controlthe pH in a part of the process, for example, as input into the decisionon the addition of either acidic or basic components. In someembodiments, the pH value is used to determine whether a process hasreached a certain stage, for instance, whether a reaction is atcompletion. In some embodiments, the pH value is used to determine theaddition of nutrients or other components to a reaction containing anorganism to maintain the health and productivity of the organism.

The process control step can be automated such that a given pHmeasurement value from the sensor results in the change of a processparameter without the intervention of a person. In other embodiments,the pH measurement is viewed by a person who uses the information tomake the decision the change of a process parameter.

The process control step can be controlled by a voltammetric pH systemwith a semiconductor electrode that has a sensor, a voltage source, acurrent measuring detector, and a computer for determining the pH fromthe current measurements. The voltammetric pH system can be incommunication either with a process control system, or with an operator,by analog or digital means, either with a wire, wireless connections,fibers or combinations thereof.

Another aspect of the invention is a method of voltammetric pH sensingwith a semiconductor electrode wherein the pH sensor requires littlecalibration. In some embodiments, the pH sensor is substantially free ofthe need for calibration or re-calibration. Substantially accuratemeasurements may be made without re-calibration.

The use of the voltammetric pH sensing with a semiconductor electrodehas a number of advantages. For example, the sensors of the presentinvention generally comprise solid-state sensors. The sensors of thepresent invention have a built-in internal standard such thatcalibration is not required. The sensors of the present invention can beconstructed to be physically robust, such that they are not prone tobreakage. The sensors of the present invention can be made to berelatively insensitive to fouling. The sensors of the invention can beconstructed to be resistant to chemical sterilization such as exposureto ethylene oxide, UV stabilization, gamma irradiation, electron beamirradiation, and temperature treatment. The sensors of the invention canbe constructed to be resistant to high humidity and high temperaturetreatment under pressure such as experienced in an autoclave.

The voltammetric pH sensing methods with a semiconductor electrodescomprise reactions carried out in stainless steel reactors, glassreactors (e.g., for product development), and disposable reactors (e.g.,plastic reagent bags), for example reactors described by manufacturerssuch as Wave Biotech, Hyclone, Xcellerex, and Stedim.

Another aspect of the invention provides methods for voltammetric pHsensing with a semiconductor electrode for processing includingchromatography and tangential flow ultrafiltration.

Another aspect of the invention is as a sensor in a remote monitoringsystem such as a drug (pharmaceutical agent) delivery system. Such asystem is described, for example, in U.S. Patent Application2003/0153900, which is entirely incorporated herein by reference. Theanalyte monitoring system or monitoring and drug (pharmaceutical agent)delivery system can be partitioned into a disposable module, a reusablemodule and a personal digital assistant (PDA) module. A PDA is typicallya portable, e.g., handheld device that has computing and networkingcapability, and a user interface, with output, e.g., a display, andinput, e.g., stylus, keyboard, and/or touchscreen capability. Thisconfiguration optimally distributes functionality among these threeconfigurations to achieve certain advantages. However the invention isnot limited to this configuration. For example, a one-unit disposabledevice including all electronics, microneedles, chemistry, sensors,mechanics and user interface may be alternatively employed. Or, morerelevantly, the design of the invention allows for any distribution ofcomponents between one or more system modules. For example, componentsmay be partitioned among one or more system modules based on the overallsystem cost, user safety and/or performance concerns.

The disposable module contains those components that once used must bediscarded to maintain safety and accuracy. This module preferablyincludes any structural or mechanical elements to maintain integrity,sterility and/or an electromechanical interface to any reusablecomponents. Therefore this system module can include, for example:microneedles, a microfluidic assembly, membrane, reagent chemistry andassociate housing materials. The portion of a sensor which is in contactwith a biological fluid, for example, may be part of the disposablemodule. This module can also include retaining mechanisms forestablishing and maintaining intimate contact with the body therebyproviding mechanical retention of the analyte monitoring/drug(pharmaceutical agent) delivery system.

The reusable module generally contains those components that control,automate motion, measure the analyte concentration, alarm the user,transmit data to the PDA module. This module can also include retainingmechanisms. Generally, this module includes: a microprocessor withassociated circuitry (e.g., memory, supporting electronics and thelike), sensing circuitry, including, for example, a voltage supply andcurrent measuring device, drive mechanisms such as motors or the like, apower supply (e.g., battery) and an interface operable to communicatewith a portable computing device or PDA. The interface can be RF,magnetic or inductive, optical or the like. The reusable module can alsohave an audible or vibration alarm to notify the user that user actionintervention is required.

The PDA module generally includes a separate user interface via aportable computing device such as a personal digital assistant (PDA),handheld computer or the like for controlling and/or interacting withthe device. A typical portable computing device includes a processor,memory, associated circuitry, a display (e.g., monochrome or color LCD)and an input device such as a key pad, touch screen (e.g., integratedwith the display) or the like and an operating system. The display canshow the value of the analyte to be measured, could provide the userwith instructions on how to respond to the measured level of analyte, ormay tell the user what automatic actions have been taken in response toa measured level of analyte.

Today, portable computing devices with improved operating systemsoftware and user interfaces are readily available. These devicesprovide the potential for rich and extended functionality. For example atypical PDA includes a relatively large viewing screen and can alsoinclude wireless communications mechanisms, a sophisticated operatingsystem and a variety of business and personal software (calendars,scheduling, etc.). The invention preferably includes the use of a PDA toprovide the proprietary software (programs) for autonomous operationwith an improved user interface.

For example, the PDA module can provide the user with software thatfacilitates informed decisions to help a patient user more optimallyadjust either drug levels or behaviors to more optimal levels. The PDAconfiguration provides a user interface and preferably allows users theability to program and or control testing. The user can view individualanalyte measurements and graphically display analyte level trends by theday, week or custom time period. The PDA can be used to display any andall of the measurements recorded by the system. Using the propersoftware, the user can be provided with recommendations for drugregiment modification. In some cases, the user can program the timeswhen their analyte tests are to be taken. Preferably, the user can alsoset the upper and lower limits for alerts.

The system can be programmed such that whenever a user makes changes andwith verification from the user, the information can be wirelesslydownloaded to the system. During the day the user may not need to usethe PDA unless alerted by the system to check for an analyte reading.The user can initiate a test from the PDA if wanting to make animmediate measurement. Once the user selects this command, verifies it,and transmits it to the reusable module, a confirmation is made back tothe PDA.

Another aspect of the invention comprises drug dispenser capsulecomprising a voltammetric sensor. In some embodiments, the drugdispenser capsule comprises a semiconductor based voltammetric sensor asdescribed herein. The drug dispenser capsule of the present inventioninternally senses a biologic condition by the detection of the presenceor amount of an analyte, and internally dispenses drugs within thedigestive tract of a body (e.g., a human body or animal body) based uponthe sensed level of analyte. The capsule is inert and is thereforeswallowable and passable through the digestive tract without beingconsumed. By sensing the level of one or more analytes, the swallowabledrug dispenser capsule senses information the digestive tract or sensesconditions within the digestive tract that are indicative of conditionsin other organs (e.g., skin). In addition to the voltammetric analytesensor, the capsule contains one or more other sensors (e.g., chemical,electrical, etc.) so that more types of biologic data can be trackedthrough the digestive system. In response to that sensed information,the capsule dispenses a bioactive substance within the digestive tractwithout the need to transmit or receive signals from a remotetransmitter/receiver, and without active human or computer management.Drug dispenser capsules are described, for example, in U.S. Pat. No.6,929,636, which is entirely incorporated herein by reference.

The swallowable drug dispensing capsule comprising a voltammetric sensorcan include, for example, sensors, a controller, memory, optionalprogrammable logic, a power supply, a microactuator, a drug storagemodule, and communication interface having at least one of the followingtypes of communication modules: radiofrequency; ultrasonic; and/orinfrared. In one preferred embodiment, at least memory, and preferablyalso controller and/or programmable logic are embodied on asemiconductor-based, e.g., silicon-based, module in one or moresemiconductor chips.

In some embodiments, the swallowable drug dispensing capsule hasmultiple sensors that are arranged an outer surface of capsule in adesired predetermined orientation that is expected to expose each sensorto a targeted bodily condition or landmark within the human body. Eachsensor can comprise a single type of sensor such as an image detector ora different type of sensor (e.g., chemical, electrical, temperature,etc.). Chemical detectors detect the presence of many analytes, such aspH, or other analytes.

The swallowable drug dispensing capsule of the invention can have acontroller that regulates communication between sensors and memory,communication between memory and any remote controllers outside of thehuman body, and communication with programmable logic component(s).Finally, controller can operably control both communication interfaceand a microactuator. The controller typically is a logic controller andincludes a microprocessor. The controller may also comprise one or morelogical devices (e.g., a logic gate) capable of performing a sequence oflogical operations.

The swallowable drug dispensing capsule generally has a memory orstorage device that is typically an ultra-high capacity storage device,and which is often based on a semiconductor chip, e.g., a silicon chip.

The swallowable drug dispensing capsule generally has a drug storagemodule and a microactuator. The drug storage module represents acontainer for holding a drug or bioactive substance that may bereleased, for example, into the digestive tract. Accordingly, the drugstorage module also includes one or more selectively activateddispensing ports that open in an outer surface of capsule. Themicroactuator can have a chemically activated or electromechanicallyactivated mechanism for causing the drug storage module to release itscontents. The swallowable drug dispensing capsule has a suitable powersupply, such as a lithium-ion battery, which is relatively non-toxic.Alternatively, other power supplies that are suitable for in vivoenvironments can be used.

The swallowable drug dispensing capsule generally has a communicationinterface that includes any suitable wireless transmission technology(e.g., ultrasonic, radiofrequency, etc.) that readily permitscommunication to and from the capsule while the capsule is in digestivetract and the remote transmitter/receiver which is located remotelyoutside of the body. However, a wireless port is preferably used forcommunicating with capsule after capsule is captured from the body.Likewise, a wireless port may be used for programming the controller,memory, and/or logic component prior to insertion of capsule within thebody to determine the manner that the sensors will operate andcommunicate with the memory, as well as the manner that microactuatorwill operate and communicate with memory via controller.

In use, the sensors, including the voltammetric sensor of the capsulesense analyte concentrations and biologic data within the digestivetract and the sensed data is passed through the controller for storagein memory and/or comparison with a stored data profile in memory and/orlogic. After the predetermined criteria are met, controller activatesmicroactuator to dispense the drug from drug storage module intodigestive tract. The sensed data optionally is stored in memory andretrieved via the communication interface after capture of capsule uponexiting the digestive tract. Finally, a wireless communication systemoptionally can be used in addition to, or as an alternative to,controller and memory to facilitate evaluating and storing sensed dataand to dispense drugs upon selective activation at the appropriate time.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used in manufacturing operations such as themanufacture of coatings, cleaners and sealers that enhance paint andfinish bonding, metal passivation to protect substrates during shipmentand storage, paint spray booth treatments that enhance quality andefficiency, and air scrubbers that limit pollutant emissions. In theseapplications, the reliable measurement of pH can be an integral part ofthe process.

In some embodiments, sensors can be used as embeddable corrosionmeasuring instruments that are capable of providing information relatedto corrosion rate, corrosion potential, conductivity and chlorideconcentration, and/or pH levels of steel rebar reinforced structures.The devices can be used to monitor the integrity of the steel. Thedevices and systems of the present invention do not require a directelectrical connection to the reinforcement steel within the structure,using the structural steel as one of the referencing materials. Sincethe disclosed instruments do not require proximity to the steel withinthe structure, the instruments can be dispersed at critical locationswithin the structure, regardless of steel placement. In someembodiments, the systems and devices are self-contained, incorporatingall required sensing electrodes and electronics. The devices can bedeployed as described in U.S. Pat. No. 6,690,182, which is entirelyincorporated herein by reference.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used in winemaking. Measurements of various propertiesincluding pH are taken throughout the process, including during (1)pressing, (2) primary fermentation, which often takes between one andtwo weeks, where yeast converts most of the sugars in the grape juiceinto ethanol (alcohol) (3) secondary fermentation.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used in brewing. The measurement of pH can be importantat the various stages of brewing, for example, at mashing, lautering,lauter tun, mash filter, boiling, whirlpool, wort cooling, fermenting,conditioning, filtering, and secondary fermentation. The semiconductorelectrode voltammetric pH sensors of the present invention can beparticularly important during fermentation, where the voltammetric pHsensors of the present invention are advantageous as they require littleto no calibration, and can be made to resist fouling during thefermentation process.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used in the production of biofuels, including theproduction of biodiesel, ethanol, butanol, and substitutes for gasoline,diesel, jet fuel, and additives to be used in any of the forgoing. Theproduction of ethanol includes both the process of converting thecellulose to sugars, and the process of converting the sugars toethanol. Although there are several key technological differences in howethanol is produced from corn or cellulosic feedstock, both paths toethanol production typically require a fermentation step that involvesthe conversion of glucose and other sugars to ethanol. Currently,baker's yeast, Saccharomyces cerevisiae, provides the primarymicrobiological system used by the corn-based ethanol industry. Themethods of the present invention relate to ethanol production for fuelfrom Saccharomyces cerevisiae and other organisms. The control of pH canbe useful in catalytic biofuel production processes, such as for theproduction of biofuel.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used in oil recovery and refining. The sensors can beincorporated into down-hole devices for measuring the analytes presentin the down-hole environment. The sensors can be used in other aspectsof processing the oil such as in oil refining.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used in the production of biopharmaceuticals, forexample, medical drugs produced using biotechnology. They include, forexample, proteins (including antibodies), nucleic acids (DNA, RNA orantisense oligonucleotides) used for therapeutic or in vivo diagnosticpurposes. Biopharmaceuticals are produced by means other than directextraction from a native (non-engineered) biological source. An exampleis recombinant human insulin (rHI, trade name Humulin), which wasdeveloped by Genentech and marketed by Eli Lilly.

Another aspect of the invention is a semiconductor electrodevoltammetric pH sensor for the production of biopharmaceuticalsincluding: blood factors (e.g., Factor VIII and Factor IX), thrombolyticagents (e.g., tissue plasminogen activator), hormones (e.g., insulin,growth hormone, gonadotrophins), haematopoietic growth factors (e.g.,erythropoietin, colony stimulating factors), interferons (e.g.,interferons-α, -β, -δ), interleukin-based products (e.g.,interleukin-2), vaccines (Hepatitis B surface antigen), monoclonalantibodies (e.g., infliximab, basiliximab, abciximab, daclizumab,gemtuzumab, alemtuzumab, rituximab, palivizumab, trastuzumab(herceptin), and etanercept) and other products such as tumor necrosisfactor, and therapeutic enzymes.

Another aspect of the invention is a method for forming a proteincomprising carrying out a fermentation reaction that produces suchprotein, wherein the pH of the fermentation reaction is controlled bymeasuring the pH with a pH sensor comprising a solid state (e.g.,semiconductor) electrode with a surface having immobilized thereon aredox active moiety that is sensitive to the presence of hydrogen ion,and using the measured pH to control the pH of the fermentationreaction. The solid state electrode in some cases is formed of asemiconductor, such as silicon. In some embodiments, the control of thepH can be manual, for instance, where an operator reads the pH from thepH sensor and uses the measured pH to determine whether or how much toadjust the pH, and in other embodiments, the control can be automatic,where the pH measurement is read by instruments that can adjust the pHbased on the value of the measurement received.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used for biopharmaceuticals produced from microbialcells (e.g., recombinant E. coli), mammalian cell lines and plant cellcultures in bioreactors of various configurations.

Cell culture requires cells to be grown, often under a strict set ofconditions to maintain the health of the cells and maximize theproduction of the culture. Cells are grown and maintained at anappropriate temperature and gas mixture (for example, 37° C., 5% CO2) ina cell incubator. Culture conditions vary widely for each cell type, andvariation of conditions for a particular cell type can result indifferent phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factorin culture systems is the growth medium. Recipes for growth media canvary in pH, glucose concentration, growth factors, and the presence ofother nutrient components. The effect of changes in pH can be dramaticin some cases, and it can be important to maintain the pH. The devices,systems, and methods of the invention allow for control of pH within arange of 1, 0.5, 0.02, 0.1, 0.05, 0.02, 0.01 pH units or less tomaintain the growth and health of the cells. The semiconductorelectrodes of the present invention allow for the accurate measurementof pH with limited fouling, and in some embodiments, no need forcalibration.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used for cells that are grown completely in solution,and for cells that are grown on a substrate. Some cells naturally livewithout attaching to a surface, such as cells that exist in thebloodstream. Others require a surface, such as most cells derived fromsolid tissues. Cells grown unattached to a surface are referred to assuspension cultures. Other adherent cultures cells can be grown ontissue culture plastic, which may be coated with extracellular matrixcomponents (e.g., collagen or fibronectin) to increase its adhesionproperties and provide other signals needed for growth.

Another aspect of the invention is a bioreactor or fermentor in whichthe reaction or fermentation occurring therein is controlled by asemiconductor based voltammetric sensor of the invention. In anembodiment, the invention comprises a bioreactor comprising asemiconductor based sensor wherein the semiconductor sensor comprises asemiconductor surface having immobilized thereon a redox active moietythat is sensitive to the presence of an analyte, such as hydrogen ion.FIG. 17 shows an example of a bioreactor of the invention comprising aprobe for measuring pH, and thereby controlling the pH in the reactorduring the reaction. The probe comprises an electrode having asemiconductor surface having immobilized thereon a redox active moietythat is sensitive to hydrogen ion. In some embodiments the probecomprises two electrodes, each comprising a semiconductor surface, oneof the electrodes having immobilized thereto a redox active moiety thatis sensitive to hydrogen ion, and one of the electrodes having attachedthereto a redox active moiety that is insensitive to hydrogen ion. Insome embodiments the probe further comprises a counter electrode, and insome embodiments it further comprises a reference electrode.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used to assist in the successful manipulation ofcultured cells. As cells generally continue to divide in culture, theygenerally grow to fill the available area or volume. This can generateseveral issues that the reliable measurement of pH can assist with, suchas: Nutrient depletion in the growth media; accumulation ofapoptotic/necrotic (dead) cells; cell-to-cell contact stimulating cellcycle arrest, causing cells to stop dividing known as contactinhibition; cell-to-cell contact stimulating promiscuous and unwantedcellular differentiation Sometimes these issues can be identified bymonitoring pH, alone or in combination with other measurements, and canthen be controlled or remediated by adjusting tissue culture conditionsthat often rely on sterile techniques. These methods aim to avoidcontamination with bacteria or yeast that will compete with mammaliancells for nutrients and/or cause cell infection and cell death. The pHmeasurements of the present invention are amenable to being carried outin a biosafety hood or laminar flow cabinet to exclude contaminatingmicro-organisms.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used for pH sensing in plant tissue culture, bacterialand yeast cell culture, and viral cell culture.

Another aspect of the invention is a semiconductor electrodevoltammetric pH sensor for sensing pH in plant tissue culture. The pHmeasurements of the present invention can be used at any step of plantcell culture. Plant tissue culture is typically performed under asepticconditions under filtered air. Living plant materials from theenvironment are naturally contaminated on their surfaces (and sometimesinteriors) with microorganisms, so surface sterilization of startingmaterials (explants) in chemical solutions (usually alcohol and mercuricchloride) is an important first step. Explants are then usually placedon the surface of a solid culture medium, but are sometimes placeddirectly into a liquid medium, particularly when cell suspensioncultures are desired. Solid and liquid media are generally composed ofinorganic salts plus a few organic nutrients, vitamins and planthormones. Solid media are prepared from liquid media with the additionof a gelling agent, usually purified agar. The pH measurements of thepresent invention can be made in the liquid, in the moist soil, or inthe agar. The composition of the medium, particularly the plant hormonesand the nitrogen source (nitrate versus ammonium salts or amino acids),and the pH, can have profound effects on the morphology of the tissuesthat grow from the initial explant. For example, an excess of auxin willoften result in a proliferation of roots, while an excess of cytokininmay yield shoots.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used with any cell line including: National CancerInstitute's cancer cell lines, zebrafish ZF4 and AB9 cells, Madin-DarbyCanine Kidney MDCK epithelial cell line, Chinese Hamster Ovary CHOcells, Insect cell line Sf21, MCF-7 (breast cancer), MDA-MB-438 (breastcancer), U87 (glioblastoma), A172 (glioma), HeLa (cervical cancer), HL60(promyelocytic leukemia), A549 (lung cancer), HEK 293 cells(kidney-original HEK line is contaminated with HeLa), SHSY5Y Humanneuroblastoma cells, cloned from a myeloma, Jurkat cell line, derivedfrom a patient with T cell leukemia, BCP-1 cells (PEL), Primate celllines, Vero (African green monkey Chlorocebus kidney epithelial cellline initiated 1962), COS-7 (African Green Monkey Kidney Cells), Rattumor cell lines, GH3 (pituitary tumor), 9L (glioblastoma), Mouse celllines, 3T3 cells, MC3T3 (embryonic calvarial), C3H-10T1/2 (embryonicmesenchymal), NIH-3T3 (embryonic fibroblast), Invertebrate cell lines,C6/36 Aedes albopictus (Asian tiger mosquito) larva, Plant cell lines,Tobacco BY-2 cells (kept as cell suspension culture, they are modelsystem of plant cell).

Another aspect of the invention is a semiconductor electrodevoltammetric pH sensor for use in water purification. Water purificationis the process of removing contaminants from a raw water source, thegoal is generally to produce water for a specific purpose with atreatment profile designed to limit the inclusion of specific materials.Water purification is not only water purified for human consumption ordrinking water. The semiconductor electrode voltammetric pH sensors ofthe present invention can also be used water purified to meet therequirements of medical, pharmacology, chemical and industrialapplications. The semiconductor electrode voltammetric pH sensors of thepresent invention can be used in water purification processes including,but not limited to ultraviolet light; filtration; water softening;reverse osmosis, ultrafiltration; molecular stripping; deionization; andcarbon treatment. Water purification may remove particulate sand,suspended particles of organic material; parasites, such as giardia;cryptosporidium; bacteria; algae; virus; fungi, etc; minerals such ascalcium, silica, magnesium; and toxic metals such as lead, copper; andchrome. Some purification may be elective in its inclusion in thepurification process; examples, smell (hydrogen sulfide remediation),taste (mineral extraction), and appearance (iron encapsulation).

Water from any source is applicable to the present invention.Groundwater (well water) is an economical choice for drinking water, asit is inherently pre-filtered, by the aquifer from which it isextracted. Water from an aquifer will have a limited output and can takethousands of years to recharge. Surface water; (rivers, lakes, streams)is far more abundant and is the typical raw water source used to makedrinking water, as a water source it is carefully monitored for thepresence of a variety of contaminants. The methods of the presentinvention encompass the voltammetric measurement of pH of these types ofwater where the pH value of the measurement can be used to decide on thepurity of the water.

The semiconductor electrode voltammetric pH sensors of the presentinvention can be used with water purification methods including: pumpingand containment, screening, storage, pre-conditioning, pre-chlorination,and removal of the fine solids, micro-organisms and some dissolvedinorganic and organic materials.

Distilled water generally has an average pH of about 7 (neither alkalinenor acidic) and seawater generally has an average pH of 8.3 (slightlyalkaline). If the water is acidic (lower than 7), lime or soda ash canbe added to raise the pH. Lime is the more common of the two additivesbecause it is cheaper, but it also adds to the resulting water hardness.Neutralizing with soda ash, however, increases the sodium content of thewater. Making the water slightly alkaline helps ensure that coagulationand flocculation processes work effectively and also helps to minimizethe risk of corrosion in pipes and pipe fittings. The pH value can beused to determine whether water is likely to be hard or soft. Ingeneral, water with a low pH (<6.5) is acidic, and tends to be soft, andcorrosive. Therefore, the water could contain metal ions, such as iron,manganese, copper, lead, and zinc. In some cases this results inelevated levels of toxic metals. This can cause premature damage tometal piping, and have associated aesthetic problems such as a metallicor sour taste, staining of laundry, and the characteristic “blue-green”staining of sinks and drains. More importantly, there are health risksassociated with these ions or contaminants. The primary way to treat theproblem of low pH water is with the use of a neutralizer. Theneutralizer feeds a basic solution into the water to prevent the waterfrom reacting with the household plumbing or contributing toelectrolytic corrosion. Water with a pH>8.5 could indicate that thewater is hard. Hard water does not pose a health risk, but can causeaesthetic problems. These problems include an alkali taste to the water,formation of a deposit on dishes, utensils, and laundry basins,difficulty in getting soaps and detergents to lather, and formation ofinsoluble precipitates on clothing.

Another aspect of the invention is sensing of analyte levels such at thepH of bodies of water for example for resource control. The body ofwater can be, for example, a lake, ocean, stream, or river. The abilityof the invention to be used remotely and to be used without the need forfrequent calibration or any calibration at all allows the systems,devices, and electrodes to be deployed remotely in bodies of water toprovide information on analytes such as hydrogen ion, etc. in suchremote bodies.

Another aspect of the invention is a semiconductor electrodevoltammetric pH sensor for the measurement of pH in processes related tosewage treatment. Sewage treatment can have the same steps as describedabove, but may refer to water that has a higher level of contamination.Raw influent (sewage) can be the liquid and semi-solid waste fromtoilets, baths, showers, kitchens, sinks etc. Household waste that isdisposed of via sewers can compose the sewage. In some areas sewage alsoincludes some liquid waste from industry and commerce.

Stability Control

In some cases, traditional electrochemical sensors can suffer from IVfluctuations during measurement, which can lead to inaccuratemeasurements and low measurement times as more measurements would haveto be taken to obtain a time-averaged measurement. Such fluctuations canlead to instability in square wave voltammetry (SWV).

In some cases, exposing a ferrocene functionalized silicon surface to analkaline environment can shift the reaction Fe²⁺

Fe³⁺+e⁻ away from equilibrium. The Fe³⁺ (ferricenium) ions can reactwith the anion in the alkaline solution and lose the ability to reverseback to Fe²⁺ (ferrocenium) ions. This can adversely affect the stabilityof an electrochemical sensor having a ferrocene-containing workingelectrode.

In some embodiments, an electrochemical sensor is provided having one ormore working electrodes comprising species that are active to an analyteof interest, the species disposed over a solid state (e.g.,semiconductor) surface, such as a silicon surface. One or more of theworking electrodes can be covered by a layer of a polymeric material. Inan embodiment, one working electrode comprises a layer of a redox-activemoiety and a layer of polymeric material over the layer of aredox-active moiety. In another embodiment, an electrochemical sensorcomprises two working electrodes with each working electrode comprisinga layer of a redox-active moiety and a layer of polymeric material overthe layer of the redox-active moiety. The electrochemical sensor canfurther include a counter electrode and a reference electrode (asdescribed above). In an embodiment, the electrochemical sensor includestwo working electrodes, a first working electrode having a redox-activemoiety that is sensitive to an analyte of interest (e.g., H+) and asecond working electrode having a redox-active moiety that isinsensitive to an analyte of interest. In an embodiment, the electrodehaving a redox-active moiety that is insensitive to an analyte comprisesa layer of a polymeric material. In some embodiments, the layer of thepolymeric material can include a homopolymer of a copolymer. In anembodiment, the layer of the polymeric material can include afluoropolymer-copolymer. In another embodiment, the layer of thepolymeric material can include a sulfonated tetrafluoroethylene basedfluoropolymer-copolymer. In another embodiment, the layer of thepolymeric material can include perfluorosulfonate inonomer or aderivative of perfluorosulfonate inonomer. In another embodiment, thelayer of the polymeric material can include Nafion, having the formulaC₇HF₁₃O₅S*C₂F₄, and the following structure:

The layer of polymeric material, such as a Nafion membrane (or film orlayer), can advantageously stabilize its response in square wavevoltammetry (SWV). This can aid in preventing peak potentials of theworking electrode from changing with pH, which could otherwise causedeterioration of sensor performance. In addition, the layer of polymericmaterial can protect the semiconductor surface of the electrochemicalsensor against changes in solution pH, which can stabilize the peakposition for this electrode in SWV. In an embodiment, in cases in whicha working electrode comprises ferrocene, the layer of polymeric materialcan selectively screen off anions and only allow the passage (or flow)of cations, which can minimize the interaction of anions and ferriceniumions, thereby preserving the integrity of the redox reaction.

In an embodiment, an electrochemical sensor is provided having a workingelectrode comprising a redox active moiety and a layer of polymericmaterial over the redox active moiety. The layer of polymeric materialcan include Nafion. The layer of polymeric material can have a thicknessbetween about 1 nanometer (“nm”) and 1000 micrometers (“microns”), orbetween about 100 nm and 500 microns, or between about 200 nm and 250microns, or between about 500 nm and 125 microns.

In an embodiment, an electrochemical sensor is provided having a workingelectrode having a redox-active moiety disposed thereon, wherein theredox-active moiety is sensitive or insensitive to an analyte ofinterest. A Nafion-containing membrane (or layer) is disposed over theworking electrode. In an example, an electrochemical sensor is providedhaving a working electrode comprising a Nafion-containing membrane overthe working electrode, the working electrode having a layer of one ormore redox-active moieties.

In an embodiment, an electrochemical sensor is provided having a workingelectrode comprising a layer of ferrocene, and a layer of a polymericmaterial over the layer of ferrocene. The layer of polymeric materialcan include Nafion. The layer of polymeric material can have a thicknessbetween about 1 nanometer (“nm”) and 1000 micrometers (“microns”), orbetween about 100 nm and 500 microns, or between about 200 nm and 250microns, or between about 500 nm and 125 microns.

In another embodiment, an electrochemical sensor is provided having aworking electrode comprising a layer of anthracene, and a layer of apolymeric material over the layer of anthracene. The layer of polymericmaterial can include Nafion. The layer of polymeric material can have athickness between about 1 nanometer (“nm”) and 1000 micrometers(“microns”), or between about 100 nm and 500 microns, or between about200 nm and 250 microns, or between about 500 nm and 125 microns.

A working electrode can include a first layer of polymeric material,such as Nafion, and a second layer of polymeric material over the firstlayer of polymeric material. The second layer of polymeric material maybe a protective layer, which may protect the first layer of polymericmaterial and the working electrode from, for example, corrosion, ordamage due to impact. In an example, a working electrode includes aNafion-containing (e.g., a porous material impregnated with Nafion) overa layer of redox-active moieties, and a layer of a second polymericmaterial over the Nafion-containing layer. In an example, the secondlayer of polymeric material includes polyethersulphone (PES).

In some cases, the second layer of polymeric material can be a lightblocking layer (see below). The light blocking layer may aid inminimizing, if not eliminating, the interaction of the working electrodewith light during operation.

In some embodiments, an electrode, such as a working electrode, cancomprise a layer of a composite material over the electrode. Thecomposite material can include a porous material, such as porous plasticor porous silica, which has been treated with a polymeric material, suchas Nafion (or other fluoropolymer-copolymer). The porous material insome cases is impregnated with Nafion. In some situations, theNafion-containing layer selectively filters out negative ions (anions)that may interfere with the operation of the electrode. In somesituations, the use of a porous material may advantageously providethickness uniformity of the Nafion layer over the electrode.

In some embodiments, the layer of the composite material, such as amembrane containing porous plastic and Nafion, has a thickness betweenabout 1 nanometer (“nm”) and 1000 micrometers (“microns”), or betweenabout 100 nm and 500 microns, or between about 200 nm and 250 microns,or between about 500 nm and 125 microns. In some embodiments, a porousmaterial used in the preparation of the composite plastic membrane (orfilm, or layer), such as porous plastic or porous silica, has one ormore pores at pore sizes (diameters) between about 1 micron and 1000microns, or between about 10 microns and 500 microns, or between about50 microns and 100 microns. In some embodiments, the Nafion content inthe pores of the porous material used in the preparation of thecomposite material, such as porous plastic or porous silica, is betweenabout 1% and 99%, or between about 10% and 50%, or between about 35% and65%. The porous composite material can be prepared by impregnation orsol-gel synthesis.

In another aspect of the invention, an electrode comprises a layer of alight blocking material that reduces, if not eliminates, the interactionof light with a surface of the electrode, such as a working electrode.Use of a light blocking layer on electrodes has led to the unexpectedrealization that device performance during device use in ambientconditions (such as upon exposure to sunlight or room light) may beimproved if incident light on a surface of a working electrode (and insome cases reference and counter electrode) is minimized, if noteliminated. The light blocking layer in some cases is covered with alayer of a protective material, such as PES. In some embodiments, anelectrode having a light blocking layer comprises a light emittingdevice (see below) on a side of the electrode opposite from the lightblocking layer. In other embodiments, the light blocking layer is formedof polyethersulphone (PES), such as an opaque PES or PES that does nottransmit light. In some cases, the PES is “black” PES. In somesituations, the light blocking layer is a porous material that isoptically opaque or otherwise blocks light from reaching a surface of anelectrode. The porous material permits an analyte to reach a surface ofthe electrode while minimizing, if not eliminating, the interaction oflight with the surface of the electrode. Examples of such light blockinglayers include polymeric materials, such dark porous plastics. In anexample, a dark porous plastic includes PES.

In some embodiments, an electrode (e.g., working electrode, referenceelectrode, counter electrode) comprises a light blocking layer forminimizing the interaction of the electrode and/or light sensitivemoieties on or over the electrode with light, such as ambient light. Thelight blocking layer may be formed of a polymeric material, such aspolyethersulphone (PES) (e.g., black PES). In some situations, a lightblocking layer may advantageously screen off light without impeding theflow of an analyte (e.g., hydrogen ions), or impeding the interaction ofthe analyte with the surface of the electrode. In some situations, thelight blocking layer can prevent (or minimize) light from inducingelectron excitation on the semiconductor surface of the workingelectrode, which may interfere with the operation of the workingelectrode.

The light blocking layer may include one or more pores for permitting ananalyte from reaching a surface of the electrode, such as a surface of aworking electrode having redox active moieties. The light blocking layermay have pores at a pore size (diameter) between about 0.1 micrometers(“microns”) and 1 micron, or 0.2 microns and 0.9 microns, or 0.45microns and 0.8 microns.

In some embodiments, the light blocking layer can prevent between about50% to about 99.9% of light, or between about 60% and 90% of light, orbetween about 70% and 80% of light from reaching a surface of anelectrode, such as a semiconductor surface that may exhibit sensitivityto light. In some cases, the light blocking layer transmits less than20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower of lightincident on the light blocking layer. The percentage of lighttransmitted through the light blocking layer is proportional to thepercentage of light that reaches a surface of an electrode.

In another aspect of the invention, electrochemical sensors are providedhaving electrodes operatively coupled to light emitting devices. In someexamples, electrodes are formed on, or are included in, light emittingdevices that are configured to generate light. Light emitting devices insome cases are light emitting diodes (LED's).

In some situations, it has been observed that electrochemical sensorsexposed to light sources of variable wavelength and/or intensity, suchas ambient light (e.g., sunlight, room light) produces unpredictable orerratic signals (e.g., noise), which may adversely affect deviceperformance. However, it has been observed that operatively couplingelectrodes (e.g., working electrodes, reference electrode and counterelectrode) to a fixed wavelength, fixed intensity light source, such asa light emitting device (also “light-emitting device” herein),unexpectedly minimizes, if not eliminates such erratic signals duringelectrochemical measurements. In some cases, exposing an electrochemicalsensor to light during use provides for improved sensor operation whenthe electrode is exposed to ambient light. This advantageously precludesthe need to operate the sensor in the dark, for example.

In some embodiments, during use, an electrode is saturated with lightfrom a light source. In other embodiments, during use, an electrode isexposed to light such as electrons and holes generated in the electrodeupon exposure to light are at least about 1%, or 10%, or 20%, or 30%, or40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95%, or 99% of themaximum concentration of electrons and holes that may be generated inthe electrode upon exposure to light.

In some embodiments, a working provided herein includes a layer of aredox active moiety adjacent a surface of the electrode, and a lightblocking layer over the layer of the redox active moiety. The lightblocking layer in some cases substantially covers the layer of the redoxactive moiety.

In some embodiments, electrochemical sensors are provided in which oneor more electrodes, such as a working electrode, is formed on a lightemitting device, such as a light emitting diode, or a plurality ofelectrodes are formed on a light emitting device. In such a case, lightfrom the light emitting device is incident on an underside of one ormore electrodes, and may in some cases propagate through at least aportion of the one or more electrodes. The ohmic contact side of the oneor more electrodes may not be configured to come in contact with asolution having an analyte (e.g., H

In other embodiments, electrodes may not be formed on light emittingdevices, but surface of electrodes (e.g., a surface of a workingelectrode having a redox active moiety) is in view of a light emittingdevice. In such a case, light from the light emitting device is incidenton the surface of the electrode configured to come in contact with asolution having an analyte.

In some embodiments, the working electrode can be operatively coupled toa light source below or above a surface of the working electrode. Lightfrom the light source may minimize, if not eliminate, interactions oflight with a surface of the working electrode. In some embodiments, thelight source comprises a lamp, a light-emitting diode (LED), or otherlight emitting device that illuminates a top surface (having redoxactive moieties) of the working electrode.

In other embodiments, the light source illuminates an underside of theworking electrode. The underside may not have redox active moieties. Insuch a case, the working electrode may be sufficiently thin to permitlight to propagate through the working electrode.

FIG. 29 shows a working electrode having a light-emitting device, suchas a light emitting diode, and an electrode adjacent to thelight-emitting device. In some embodiments, the light-emitting device isa light emitting diode (LED) having an active material (or activeregion) configured to generate light upon the recombination of electronsand holes. In some embodiments, the working electrode is formed of asemiconducting material, such as silicon. The working electrode can beformed over the LED layer using, for example, spincoating or vapordeposition techniques, such as chemical vapor deposition or atomic layerdeposition. The surface of the working electrode further comprises aredox-active moiety immobilized thereon, said redox-active moietyexhibiting an oxidation potential and/or a reduction potential that issensitive or insensitive to the presence of an analyte. In someembodiments, the redox-active moiety can be immobilized on thesemiconducting layer by reactions such as hydrosilylation, free radicalreactions, carbodiimide coupling, Diels-Alder reactions, Michaeladdition, epoxy reactions, or click chemistry.

In some embodiments, the working electrode comprises anon-semiconducting material, such as carbon. In some embodiments, alayer of non-semiconducting material can be combined with the LED layerby, for example, spraycoating or vapor deposition techniques. Thesurface of the working electrode further comprises a redox-active moietyimmobilized thereon, said redox-active moiety exhibiting an oxidationpotential and/or a reduction potential that is sensitive or insensitiveto the presence of an analyte. In some embodiments, the redox-activemoiety can be immobilized on the non-semiconducting layer, such asactivated carbon, by first oxidatively treating the non-semiconductinglayer to create oxygen groups on the layer (Lemus-Yegres et al.,Microporous and Mesoporous Materials 109 (2008), 305-316). Thenon-semiconducting layer can then be subjected to immobilizationreactions.

In some cases, the LED may minimize, if not eliminate, interactions oflight with the surface of the working electrode.

In an example, a working electrode includes a light emitting diodehaving a p-type Group III-V semiconductor material, an active layer(e.g., multiple quantum well active layer adjacent to the p-type GroupIII-V semiconductor material), and an n-type Group III-V semiconductormaterial layer adjacent to the active layer. A first electrode is formedadjacent to the p-type Group III-V semiconductor material. A secondelectrode is formed adjacent to the n-type Group III-V semiconductormaterial layer. The second electrode may be formed over the n-type GroupIII-V semiconductor material layer with the aid of a transition layer,such as indium tin oxide. The electrode may be formed of one or moretransition metals, such as gold and/or silver. A layer of an insulatingmaterial is then formed on the electrode. In some cases, the layer ofthe insulating material is formed of a metal oxide or a nitride (e.g.,silicon nitride). Next, a third electrode is formed on the layer of theinsulating material. The third electrode will serve as the back contactto the working electrode. Next, the working electrode is formed on thethird electrode. Redox active moieties are then provided on a surface ofthe working electrode. The working electrode may be formed of asemiconducting (e.g., silicon, germanium, gallium nitride) ornon-semiconducting (e.g., carbon, a metal) material.

In some embodiments, a light emitting device adjacent an electrode, suchas a working electrode, emits light having a wavelength greater than orequal to about 300 nanometers (nm), 400 nm, 500 nm, 600 nm, 700 nm, or800 nm. In some situations, the light emitting device emits light havinga wavelength above 750 nm or 800 nm. In an example, the light emittingdevice emits near infrared or infrared light.

Light may be exposed to sensor surfaces having redox active moieties, orsensor surfaces opposite from the redox active moieties (e.g., backsurfaces of a sensor). In some cases, the thickness of a workingelectrode is selected to provide a desirable sensor output when exposedto light from the backside. In some embodiments, a working electrode hasa thickness between about 100 nanometers (“nm”) and 1 millimeter, orbetween about 500 nm and 750 micrometers (“microns”), or between about250 microns and 650 microns.

In some embodiments, exposure of a working electrode to light, such as,e.g., a fixed intensity, fixed wavelength light, provides improvedsensor signal to noise. In some cases, the signal to noise is improvedby a factor of 10, 100, 1000, 10,000, 100,000, or more.

The wavelength of light emitted by the light emitting device, in somecases, is adjustable. In some cases, the wavelength of light emitted bythe light emitting device is adjusted based on the power applied to thelight emitting device. In some cases, the sensor has an ambient lightsensor that detects the level of ambient light, and adjusts thewavelength of light emitted by the light emitting device. In an example,if the ambient light sensor detects little to no ambient light, thelight emitting device will not be used. In another example, if theambient light sensor detects light above a predetermined threshold, thenthe light emitting device may be used.

Light emitting devices provided herein may be controlled with the aid ofprocessors and software. The processor may be an on-board processor,such as mounted on a printed circuit board in proximity to the sensorand any ambient light sensor. The processor may aid in regulating thefunctionality of any of the sensor, the light emitting device, and theambient light sensor.

Co-Functionalization of Working Electrodes

In another aspect of the invention, an electrochemical sensor isprovided having co-functionalized working electrodes.Co-functionalization can permit the use of hydrocarbon molecules asspace fillers to cover sites on a silicon hydride (—Si—H) surface thathave not been occupied by redox active moieties, such as anthraceneand/or ferrocene. The hydrocarbon molecules can block (or passivate)such sites, thereby preventing such sites from interacting with anionsand cations during operation of the electrochemical sensor, which canprovide for improved device performance.

In an embodiment, an electrochemical sensor comprises one or moreworking electrodes, a counter electrode and a reference electrode,wherein at least one of the one or more working electrodes isco-functionalized with redox-active moieties and hydrocarbon molecules.In an embodiment, an electrochemical sensor includes a workingelectrode, a counter electrode and a reference electrode. The workingelectrode can include a mixed monolayer of redox active species, such asanthracene and ferrocene, and hydrocarbon molecules, such as alkanes,alkenes, or alkynes. In an embodiment, the hydrocarbon is a long-chainhydrocarbon. In another embodiment, the hydrocarbon comprises a chain of2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more carbon atoms. In an embodiment, the hydrocarbonmolecules can include a decyne having the following structural formula:

In an embodiment, a working electrode comprises a submonolayer coverageredox-active species, with the remaining sites (i.e., S—H sites) coveredby a hydrocarbon, such as decyne. In another embodiment, a workingelectrode can include a mixed layer of ferrocene and a hydrocarbon, suchas decyne. In another embodiment, a working electrode can include amixed layer of anthracene and a hydrocarbon, such as decyne. In anotherembodiment, a working electrode can include a mixed layer of ferrocene,anthracene, and a hydrocarbon, such as decyne.

Single-Use or Disposable Electrochemical Sensors

In another aspect of the invention, single-use and/or disposableelectrochemical sensors are provided. In an embodiment, electrochemicalsensors are provided that are suitable for single use applications,disposable applications, or both single-use and disposable applications.

In an embodiment, a disposable electrochemical sensor is provided. Inanother embodiment, a single-use electrochemical sensor is provided. Inanother embodiment, a disposable and single-use electrochemical sensoris provided.

In an embodiment, a single-use electrochemical sensor comprises anonboard energy storage device. Such an energy storage device can beconfigured to store electrical potential energy (e.g., in the form ofelectrons in excited states) and discharge upon use of theelectrochemical sensor. Energy storage devices can be selected frombatteries, capacitors, or photovoltaic modules. In an embodiment, anelectrochemical sensor comprises a battery, such as, e.g., a lithium ionbattery or nickel metal cadmium battery. In another embodiment, anelectrochemical sensor comprises a capacitor. In another embodiment, anelectrochemical sensor comprises a battery electrically coupled to acapacitor.

In another embodiment, an electrochemical sensor is provided having areplaceable energy storage device, such as a replaceable battery. Insuch a case, the electrochemical device can be used for a durationdetermined by the lifetime of the energy storage device. The energystorage device can be subsequently changed, thereby permitting furtheruse of the electrochemical sensor. In another embodiment, anelectrochemical sensor having a rechargeable energy storage device isprovided. The rechargeable energy storage device can include arechargeable battery.

In an embodiment, an electrochemical sensor, such as any electrochemicalsensor provided herein (e.g., a pH sensor), can include an energystorage device configured to provide charge to the electrochemicalsensor. In another embodiment, an electrochemical sensor includes anenergy storage device configured to provide charge for up to about 1day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days,or 8 days, or 9 days, or 10 days, or 11 days, or 12 days, or 13 days, or14 days, or 15 days, or 16 days, or 17 days, or 18 days, or 19 days, or20 days, or 21 days, or 22 days, or 23 days, or 24 days, or 25 days, or26 days, or 27 days, or 28 days, or 29 days, or 30 days, or 35 days, or40 days, or 45 days, or 50 days, or 100 days, or 150 days, or 200 days,or 300 days, or 400 days, or 500 days, or 1000 days. In anotherembodiment, an electrochemical sensor includes an energy storage deviceconfigured to provide charge for at least about 1 day, or 2 days, or 3days, or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days,or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days,or 16 days, or 17 days, or 18 days, or 19 days, or 20 days, or 21 days,or 22 days, or 23 days, or 24 days, or 25 days, or 26 days, or 27 days,or 28 days, or 29 days, or 30 days, or 35 days, or 40 days, or 45 days,or 50 days, or 100 days, or 150 days, or 200 days, or 300 days, or 400days, or 500 days, or 1000 days.

In an embodiment, a semiconductor-based electrochemical sensor isprovided having redox-active moieties. The sensor can include oneredox-active moiety that is sensitive to an analyte (e.g., hydrogenions) and another redox-active moiety that is insensitive to theanalyte. The semiconductor-based electrochemical sensor can furtherinclude an on-board energy storage device configured to provide power tothe semiconductor-based electrochemical sensor. In an embodiment, theenergy storage device can provide power to the semiconductor-basedelectrochemical sensor for a time period of at least about 1 second, or10 seconds, or 30 seconds, or 1 minute, or 2 minutes, or 3 minutes, or 4minutes, or 5 minutes, or 6 minutes, or 7 minutes, or 8 minutes, or 9minutes, or 10 minutes, or 30 minutes, or 1 hour, or 2 hours, or 3 ours,or 4 hours, or 5 hours, or 6 hours, or 7 hours, or 8 hours, or 9 hours,or 10 hours, or 11 hours, or 12 hours, or 1 day, or 2 days, or 3 days,or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or 10days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16days, or 17 days, or 18 days, or 19 days, or 20 days, or 21 days, or 22days, or 23 days, or 24 days, or 25 days, or 26 days, or 27 days, or 28days, or 29 days, or 30 days, or 35 days, or 40 days, or 45 days, or 50days, or 100 days, or 150 days, or 200 days, or 300 days, or 1 year, or2 years, or 3 years, or 4 years, or 5 years, or 6 years, or 7 years, or8 years, or 9 years, or 10 years, or 15 years, or 20 years.

In an embodiment, a semiconductor-based electrochemical sensor isprovided that is configured for single-use operation. In anotherembodiment, an electrochemical sensor is provided that is configured tooperate, upon first use, for a time period of at least about 1 second,or 10 seconds, or 30 seconds, or 1 minute, or 2 minutes, or 3 minutes,or 4 minutes, or 5 minutes, or 6 minutes, or 7 minutes, or 8 minutes, or9 minutes, or 10 minutes, or 30 minutes, or 1 hour, or 2 hours, or 3ours, or 4 hours, or 5 hours, or 6 hours, or 7 hours, or 8 hours, or 9hours, or 10 hours, or 11 hours, or 12 hours, or 1 day, or 2 days, or 3days, or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days,or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days,or 16 days, or 17 days, or 18 days, or 19 days, or 20 days, or 21 days,or 22 days, or 23 days, or 24 days, or 25 days, or 26 days, or 27 days,or 28 days, or 29 days, or 30 days, or 35 days, or 40 days, or 45 days,or 50 days, or 100 days, or 150 days, or 200 days, or 300 days, or 1year, or 2 years, or 3 years, or 4 years, or 5 years, or 6 years, or 7years, or 8 years, or 9 years, or 10 years, or 15 years, or 20 years. Inanother embodiment, after first use, the electrochemical sensor will notfunction (i.e., the electrochemical sensor will not function beyond thefirst use).

In another embodiment, an electrochemical sensor comprising one or moreworking electrodes, a counter electrode and a reference electrode isdisposed in a high density polyethylene support that can be directlywelded to a surface a bag, such as a polyethylene bag, for single usebag application. Such electrochemical sensors can be configured for usewith cell fermentation, media storage, buffer preparation and cellstorage.

In an embodiment, a redox-active moiety-containing analyte sensor isprovided that is configured for use in a time period of about 1 day, or5 days, or 10 days, or 20 days, or 25 days, or 30 days, or 1 month, or 2months, or 3 months, or 4 months, or 5 months, or 6 months, or 7 months,or 8 months, or 9 months, or 10 months, or 11 months, or 1 year, or 2years, or 3 years, or 4 years, or 5 years, or 6 years, or 7 years, or 8years, or 9 years, or 10 years. In another embodiment, a redox-activemoiety-containing analyte sensor is provided that is configured for usein a time period of at least about 1 day, or days, or 10 days, or 20days, or 25 days, or 30 days, or 1 month, or 2 months, or 3 months, or 4months, or 5 months, or 6 months, or 7 months, or 8 months, or 9 months,or 10 months, or 11 months, or 1 year, or 2 years, or 3 years, or 4years, or 5 years, or 6 years, or 7 years, or 8 years, or 9 years, or 10years. Such analyte sensors can be configured for use within aparticular time period without re-calibration.

Analyte sensors provided herein can have various sensitivities. In anembodiment, a redox-active moiety-containing analyte sensor is providedhaving a sensitivity between about 5 mV per pH unit and 200 mV per pHunit, or between about 10 mV per pH unit and 100 mV per pH unit, orbetween about 20 mV per pH unit and 60 mV per pH unit.

Analyte sensors provided herein can have various shelf lives. In anembodiment, a redox-active moiety-containing analyte sensor is providedhaving a shelf life between about 1 month and 20 years, or between about2 months and 10 years, or between about 3 months and 3 years. Suchanalyte sensors can function without re-calibration. In anotherembodiment, a redox-active moiety-containing analyte sensor is providedhaving a shelf life of at least about 1 month, or 2 months, or 3 months,or 4 months, or 5 months, or 6 months, or 7 months, or 8 months, or 9months, or 10 months, or 11 months, or 1 year, or 2 years, or 3 years,or 4 years, or 5 years, or 6 years, or 7 years, or 8 years, or 9 years,or 10 years.

Analyte sensors provided herein can have various accuracies followingremoval from storage. In an embodiment, a sensor for detecting ananalyte is provided having an accuracy to within 0.1% (pH units) whilein use or storage for at least 2 years. In another embodiment, a sensorfor detecting an analyte is provided having an accuracy to within 0.1%(pH units) while in use or storage for at least 4 years. In anotherembodiment, a sensor for detecting an analyte is provided having anaccuracy to within 0.1% (pH units) while in use or storage for at least8 years. In another embodiment, a sensor for detecting an analyte isprovided having an accuracy to within 0.1% (pH units) while in use orstorage for at least 10 years. In some embodiments, sensors can retaintheir accuracy while in storage without the need for calibration ofre-calibration. Such sensors can include redox-active moieties, asdescribed herein.

In an embodiment, a sensor for detecting an analyte is provided havingan accuracy of plus or minus 0.0001 pH units, or 0.0002 pH units, or0.0003 pH units, or 0.0004 pH units, or 0.0005 pH units, or 0.0006 pHunits, or 0.0007 pH units, or 0.0008 pH units, or 0.0009 pH units, or0.001 pH units, or 0.002 pH units, or 0.003 pH units, or 0.004 pH units,or 0.005 pH units, or 0.006 pH units, or 0.007 pH units, or 0.008 pHunits, or 0.009 pH units, or 0.01 pH units, or 0.02 pH units, or 0.03 pHunits, or 0.04 pH units, or 0.05 pH units, or 0.06 pH units, or 0.07 pHunits, or 0.08 pH units, or 0.09 pH units, or 0.1 pH units. Such asensor can include redox-active moieties, as described herein.

In an embodiment, a sensor for detecting an analyte is provided havingan accuracy to within 0.001 pH units while in use or storage for atleast 2 years. In another embodiment, a sensor for detecting an analyteis provided having an accuracy to within 0.001 pH units while in use orstorage for at least 4 years. In another embodiment, a sensor fordetecting an analyte is provided having an accuracy to within 0.001 pHunits while in use or storage for at least 8 years. In anotherembodiment, a sensor for detecting an analyte is provided having anaccuracy to within 0.001 pH units while in use or storage for at least10 years. In some embodiments, sensors can retain their accuracy whilein storage without the need for calibration of re-calibration. Suchsensors can include redox-active moieties, as described herein.

In an embodiment, a sensor for detecting an analyte is provided havingan accuracy to within 0.0001, or 0.001, or 0.01, or 0.1 pH units whilein use or storage for at least 1 year. In another embodiment, a sensorfor detecting an analyte is provided having an accuracy to within0.0001, or 0.001, or 0.01, or 0.1 pH units while in use or storage forat least 2 years. In another embodiment, a sensor for detecting ananalyte is provided having an accuracy to within 0.0001, or 0.001, or0.01, or 0.1 pH units while in use or storage for at least 4 years. Inanother embodiment, a sensor for detecting an analyte is provided havingan accuracy to within 0.0001, or 0.001, or 0.01, or 0.1 pH units whilein use or storage for at least 8 years. In another embodiment, a sensorfor detecting an analyte is provided having an accuracy to within0.0001, or 0.001, or 0.01, or 0.1 pH units while in use or storage forat least 10 years. Such sensors can include redox-active moieties, asdescribed herein.

In an embodiment, a redox-active moiety-containing analyte sensor isprovided having a shelf life of at least about 1 month, or 2 months, or3 months, or 4 months, or 5 months, or 6 months, or 7 months, or 8months, or 9 months, or 10 months, or 11 months, or 1 year, or 2 years,or 3 years, or 4 years, or 5 years, or 6 years, or 7 years, or 8 years,or 9 years, or 10 years, and an accuracy to within about 0.01%, or0.02%, or 0.03%, or 0.04%, or 0.05%, or 0.1%, or 0.15%, or 0.2%. Inanother embodiment, a redox-active moiety-containing analyte sensor isprovided having a shelf life of at least about 1 month, or 2 months, or3 months, or 4 months, or 5 months, or 6 months, or 7 months, or 8months, or 9 months, or 10 months, or 11 months, or 1 year, or 2 years,or 3 years, or 4 years, or 5 years, or 6 years, or 7 years, or 8 years,or 9 years, or 10 years, and an accuracy to within about 0.0001 pHunits, or 0.0002 pH units, or 0.0003 pH units, or 0.0004 pH units, or0.0005 pH units, or 0.0006 pH units, or 0.0007 pH units, or 0.0008 pHunits, or 0.0009 pH units, or 0.001 pH units, or 0.002 pH units, or0.003 pH units, or 0.004 pH units, or 0.005 pH units, or 0.006 pH units,or 0.007 pH units, or 0.008 pH units, or 0.009 pH units, or 0.01 pHunits, or 0.02 pH units, or 0.03 pH units, or 0.04 pH units, or 0.05 pHunits, or 0.06 pH units, or 0.07 pH units, or 0.08 pH units, or 0.09 pHunits, or 0.1 pH units. Such sensors can include redox-active moieties,as described herein.

In an embodiment, a redox-active moiety-containing analyte sensor isprovided that can be used to measure the presence or absence (orconcentration) or an analyte, without re-calibration, for at least about1 day, or 1 week, or 2 weeks, or 1 month, or 2 months, or 3 months, or 4months, or 5 months, or 6 months, or 7 months, or 8 months, or 9 months,or 10 months, or 11 months, or 1 year, or 2 years, or 3 years, or 4years, or 5 years, or 6 years, or 7 years, or 8 years, or 9 years, or 10years, at an accuracy to within about 0.01%, or 0.02%, or 0.03%, or0.04%, or 0.05%, or 0.1%, or 0.15%, or 0.2%. In some cases, the accuracyis to within about 10%, or 5%, or 1%, or 0.1%, or 0.01%, or 0.01%, or0.001%. In another embodiment, a redox-active moiety-containing analytesensor is provided that can be used to measure the presence or absence(or concentration) of an analyte, without re-calibration, for at leastabout 1 day, or 1 week, or 2 weeks, or 1 month, or 2 months, or 3months, or 4 months, or 5 months, or 6 months, or 7 months, or 8 months,or 9 months, or 10 months, or 11 months, or 1 year, or 2 years, or 3years, or 4 years, or 5 years, or 6 years, or 7 years, or 8 years, or 9years, or 10 years, and an accuracy to within about 0.0001 pH units, or0.0002 pH units, or 0.0003 pH units, or 0.0004 pH units, or 0.0005 pHunits, or 0.0006 pH units, or 0.0007 pH units, or 0.0008 pH units, or0.0009 pH units, or 0.001 pH units, or 0.002 pH units, or 0.003 pHunits, or 0.004 pH units, or 0.005 pH units, or 0.006 pH units, or 0.007pH units, or 0.008 pH units, or 0.009 pH units, or 0.01 pH units, or0.02 pH units, or 0.03 pH units, or 0.04 pH units, or 0.05 pH units, or0.06 pH units, or 0.07 pH units, or 0.08 pH units, or 0.09 pH units, or0.1 pH units. Such sensors can include redox-active moieties, asdescribed herein.

In some embodiments, a method for detecting the presence or absence ofan analyte, comprises bringing an analyte sensor in contact with asample, said analyte sensor having an electrode having immobilizedthereon a redox-active moiety, wherein the redox-active moiety exhibitsan oxidation potential and/or a reduction potential that is sensitive tothe presence of said analyte. Next, with the aid of the analyte sensor,the analyte is detected to an accuracy of within about 20% withoutre-calibration for a period of at least about 1 day. In some cases theaccuracy can be to within about 10%, or 5%, or 1%, or 0.1%, or 0.01%, or0.01%, or 0.001%. In some embodiments, the analyte sensor can detect thepresence or absence of the analyte without re-calibration for a timeperiod of at least about 1 day, or 1 week, or 2 weeks, or 1 month, or 2months, or 3 months, or 4 months, or 5 months, or 6 months, or 7 months,or 8 months, or 9 months, or 10 months, or 11 months, or 1 year, or 2years, or 3 years, or 4 years, or 5 years, or 6 years, or 7 years, or 8years, or 9 years, or 10 years.

Electrochemical Sensors with Multiple Sensor Modules

In another aspect of the invention, electrochemical sensors are providedhaving a plurality of sensor modules, each sensor module configured tomeasure an analyte of interest. In an embodiment, an electrochemicalsensor comprises at least 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9,or 10, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 100modules. In another embodiment, an electrochemical sensor comprises aplurality of modules. The modules can be configured to detect (ormeasure) the presence of the same analyte (e.g., H+) or differentanalytes (e.g., H+ and O₂).

With reference to FIG. 22A, an electrochemical sensor having threemodules, wherein each module is configured to sense an analyte ofinterest, is illustrated, in accordance with an embodiment of theinvention. The electrochemical sensor comprises a probe body that can becylindrical in shape. FIG. 22B is an enlarged view of a portion of theelectrochemical sensor of FIG. 22A, illustrating three modulesconfigured to sense (or measure the presence of) one or more analytes ofinterest. Each of the three modules can be configured to sense the sameanalyte (e.g., H+) or different analytes. For example, a first modulecan be configured to sense (and measure the concentration of) H+, asecond module can be configured to sense O₂, and a third module can beconfigured to sense NH₃. Such “multi-modal” sensor can be advantageousfor use in chemical reactors and bioreactors, where the measurement ofmultiple analytes is desirable.

Electrochemical Sensors Formed on Printed Circuit Boards

In another aspect of the invention, electrochemical sensors formed oncircuit boards are provided. With reference to FIG. 23, In anembodiment, a printed circuit board is provided. A dam can be placed ona field that fits around the printed circuit board. The field provides asupport surface that can be free of contaminants. The dam can constrainthe area between the field and the printed circuit board. A fillermaterial (“filler”) can be injected through holes in the printed circuitboard. The filler can hold the electrodes in place and exclude fluidsfrom touching the back side of the electrodes and the traces of theprinted circuit board. The filler can then be cured. Following curing,the sensor can be removed from the field and mounted into an appropriateholder, such as a probe body (see FIG. 22A).

Sensors formed on printed circuit boards can be mounted in a wide arrayof form factors. For example, they can be placed in high densitypolyethylene hubs and welded into disposable polyethylene containers. Asanother example, they can be mounted within tube walls or at theentrance and egress of columns. Such tubes walls or columns can beconfigured for use with containers having dimensions (e.g., diameters,lengths) configured for use with glass probe electrochemical (e.g., pHmeasurement) systems.

With reference to FIG. 24, In an embodiment, a sensor formed on aprinted circuit board can be mounted on a head assembly. Such headassembly can be configured to be mounted on a tube assembly or column,such as the tip of a tube assembly or column. In another embodiment, ahead assembly can be removably mounted to a tube assembly or column. Inanother embodiment, a head assembly can be irremovably mounted to a tubeassembly or column. In another embodiment, a head assembly can bemounted to a top portion of a tube or column for insertion into asolution having an analyte of interest. A protective material is appliedaround the head assembly to prevent the solution from entering a backportion of the head assembly. In an embodiment, the protective materialincludes one or more of polyurethane and an epoxy (e.g., polyexpoxide).

With continued reference to FIG. 24, the sensor includes a surfaceconfigured to come in contact with a solution having one or moreanalytes. The sensor includes a counter electrode (top) and a referenceelectrode (center). The sensor further includes working electrodes(bottom left and bottom right). One working electrode comprises asemiconductor surface that has immobilized thereon a redox active moietywhose oxidation potential and/or reduction potential is sensitive to thepresence of the analyte, and a second working electrode comprises redoxactive moiety whose oxidation potential and/or reduction potential isinsensitive to the presence of the analyte (see above).

Electrochemical Sensors for Use with Containers and Flow-ThroughContainers

In another aspect of the invention, an electrochemical sensor, such asany sensor provided herein can be used with containers and reactors suchas disposable containers and reactors. In an embodiment, anelectrochemical sensor is provided for use with a disposableflow-through container. In another embodiment, an electrochemical sensoris provided for use with a disposable reactor. In another embodiment, anelectrochemical sensor is provided for use with a disposable plug flowreactor. In another embodiment, an electrochemical sensor is providedfor use with a disposable continuous stirred tank reactor (CSTR).

In some embodiments, any electrochemical sensor (“sensor”) providedherein can be configured for use with a container, including a flexiblecontainer. In an embodiment, a sensor is provided configured for usewith a disposable flexible container. Such flexible container can havewalls formed of a polymeric material.

With reference to FIG. 25A, an electrochemical sensor is shown mountedon a wall of a container, in accordance with an embodiment of theinvention. The container can be a disposable container. Theelectrochemical sensor comprises a printed circuit board having thereona counter electrode, a reference electrode and one or more workingelectrode (one working electrode illustrated in the cross-sectionalcut-away). The printed circuit board brings the electrodes in electricalcommunication with connectors, which can be used to interface with acomputer system or device for use with the electrochemical sensor. Theelectrochemical sensor is mounted on a holder flange, which is separatedfrom the printed circuit board through a separation member, such as adamn-to-holder ring (“O-Ring”, as illustrated). The probe holder canseal to the dam. The flange of the holder can be substantially thin sothat it can be welded to a container, such as a bag. In an embodiment, aportion of the container is mounted to the holder flange with the aid ofan adhesive, such as, for example, one or more of polyurethane and anepoxy (e.g., polyepoxide). In another embodiment, a portion of thecontainer is welded to the holder flange. In another embodiment, theelectrochemical sensor can be sterilized, such as with the aid ofsterilizing chemicals, ultraviolet light irradiation, or plasmatreatment.

With reference to FIG. 25B, an electrochemical sensor is shown formed ona printer circuit board (PCB). The electrochemical sensor furtherincludes a connector for providing a connection between theelectrochemical sensor and a reader or other electronics unit, such as avoltage sync (see FIGS. 27A and 27B). The electrodes of theelectrochemical sensor are disposed at a bottom portion of the PCB. Inan embodiment, the electrodes of the electrochemical sensor, includingworking electrodes, a counter electrode and reference electrode, areformed in the PCB.

In an embodiment, the PCB can be formed of a military standard PCB board(e.g., FR4 substrate) or any PC board constructed from a non-conductivematerial, with conductive traces. The boards can be multilayered.

In various embodiments, solutions to seal or hermetically seal (also“sealing solutions” herein) sensor components are provided. In anembodiment, sealing solutions can comply with biological manufacturingstandards, such as reducing, if not eliminating, outgassing, andavoiding the use of environmentally hazardous or toxic materials. In anembodiment, a sealing solution is provided to a sensor through thepotting technique, or the application of a liquid adhesive to preventwater and other liquids from entering the sensor and causing electricalshort circuits. In an embodiment, a PCB having sensor components ismounted to a tube. An interior portion of the tube is sealed from theexternal environment with the aid of a sealing solution around the PCB.

In another aspect of the invention, electrochemical sensors are providedfor use with fluid flow channels, such as flow-through tubes or pipes.With reference to FIG. 26, an electrochemical sensor, such as the sensordiscussed in the context of FIG. 25A, is mounted in a chamber of aflow-through tube. The chamber can be configured for mating with aflange portion of the electrochemical sensor. In an embodiment, theelectrochemical sensor can be welded to the chamber. In anotherembodiment, the electrochemical sensor can be attached to the chamberwith the aid of an epoxy, for example. In another embodiment, theelectrochemical sensor can be removably attached to the chamber. Inanother embodiment, the electrochemical sensor can be irremovablyattached to the chamber.

Electrochemical Sensor Electrical Components

In some embodiments, the sensor or holder can have on-board electronicmemory, such as Electrically Erasable Programmable Read-Only Memory(“EEPROM”). The EEPROM can hold or store various items (or information)that can pair the electrochemical sensor with drive electronics. In anembodiment, the EEPROM can hold a serial number of the electrochemicalsensor. In another embodiment, probe calibration parameters (e.g., probecalibration constants) can be held in the memory of the EEPROM. Inanother embodiment, the algorithm details of the electrochemical sensorcan be held in the EEPROM.

In an embodiment, the EEPROM is provided on a printed circuit boardhaving the various electrodes of the electrochemical sensor (e.g., pHsensor). In another embodiment, the EEPROM is provided adjacent aprinted circuit board or other device having the various electrodes ofthe electrochemical sensor.

An electrochemical sensor comprising an on-board EEPROM can have variousadvantages and benefits over other electrochemical sensors. For example,an electrochemical sensor having an on-board EEPROM can be electricallyoptimized for different uses or markets. As an example a probe can beoptimized for pH near pH 7 and only scan for a small region around pH 7.An identical probe could be optimized for pH 4 and only scan an areaaround pH 4. A sensor optimized for pH 4 measurement and a sensoroptimized for pH 7 measurements can be similar, if not identical in formand function, different only the algorithm provided in the EEPROM ofeach sensor.

An EEPROM on-board an electrochemical sensor can save or hold one ormore types of information. In an embodiment, the EEPROM can hold acounter for measuring (and storing) the length time an electrochemicaldevice has been used. In another embodiment, if an electrochemicalsensor is configured for use over a certain time period, the counter canenable the electrochemical sensor to stop functioning after the timeperiod has been reached. The EEPROM can be used in conjunction with (orto facilitate) single-use functionality, as described in certainembodiments (see above). For example, if the electrochemical sensor isconfigured for use over the period of 28 days, the electrochemicalsensor can stop functioning once the 28-day time period has elapsed. Inanother embodiment, the counter can enable a user to know how much timethe user has remaining to use the sensor. In an embodiment, as thesensor is used, a write once area in the EEPROM can be incremented, andwhen this area of the EEPROM is filled, it will signal that the usefullife of the sensor is finished, and the sensor will terminate use. Inanother embodiment, once the useful life of the sensor has finished, theuser may be prevented from using the sensor.

In another embodiment, the EEPROM can transmit its serial number orother identifying information, in addition to the time remaining for theelectrochemical sensor, to a data recorder that can monitor one or moresensors in a user's operation.

Alternatively, sensor calibration parameters can be stored on a remotecomputer system (e.g., the “cloud”), such as a server, and transmittedto a system in electrical communication with the sensor.

In some situations, a sensor can include an identification member, suchas radio-frequency identification (RFID) tag, to enable a systemcommunicating with the sensor to identify the sensor and determine, forexample, any calibration parameters that may be required by the sensor.The identification member can be on a body or housing of the sensor.Calibration parameters may be specific to a particular application(e.g., pH measurements) of the sensor. The identification member canenable the system to provide proper calibration parameters, as may berequired for a particular sensor application.

Electronics Components for Integration into Third-Party Systems

In another aspect of the invention, an electronics component is providedfor emulating various features of current third-party or traditionalelectrochemical sensors. Such electronics components can advantageouslypermit the integration of electrochemical sensors provided herein intovarious current or traditional systems, such as glass probe systems.

Many laboratories and manufacturing facilities have existing third partypH meters, either stand alone or within digital control units. In someembodiments, an electronics unit is provided for integration intoexisting third party pH meters and systems, such as analog pH meters andsystems. In an embodiment, the electronics unit can do one or more ofemulating the functionality of a potentiostat, providing power theelectrochemical sensor, and communicating with a third party pH meter toprovide the user with a direct pH readout.

In an embodiment, the electronics unit enables communication between athird-party reader (or meter) and any probe provided herein. Theelectronics unit can further provide power to the probe. In anembodiment, the electronics unit includes a power input for providingpower to one or more of the electronics unit and a probe in electricalcommunication with the electronics unit, an input from the probe, and anoutput to a reader.

With reference to FIG. 27A, an electronic unit is shown, in accordancewith an embodiment of the invention. The electronic unit, which isillustrated as an electrical box, can permit integration ofelectrochemical sensors provided herein into systems, includingelectronics components, configured for use with types of electrochemicalsystems. With continued reference to FIG. 27A, the electrical box can beconnected to a sensor on one side and a traditional readout unit ordevice on the other side. In at least some embodiments, electrochemicalsensors are provided that use microprocessors to control transmitterelectronics to scan the sensor appropriately. The electronics componentevaluates the sensor signals and determines the appropriate sensor value(pH value in this case). The electronics component then takes the sensorvalue and computes the appropriate output voltage or current to emulatethe traditional sensor. The electronics component will then transmit theappropriate output voltage to a system or device configured for use witha traditional sensor, such as a glass probe sensor.

In some embodiments, the electronic unit of FIG. 27A can transform adigital signal into a signal that is readable by a third-party system,such as a system configured for use with a glass electrode. In anembodiment, the electronic unit can emulate the glass electrode output.The electronic unit can advantageously make sensors provided hereincompatible with all or substantially all third party electrochemicalsensors, including pH sensors.

In an embodiment, an electronics unit is provided for integratingsemiconductor-based electrochemical sensors, such as those providedherein, in devices configured for use with traditional probes, such asglass probes (e.g., glass pH probes). In another embodiment, anelectronics unit is provided that is a digital to analog converter. Suchan electronics unit can be configured to enable electrochemical sensorsof embodiments to be used with traditional or current analog probesystems.

Traditional pH meters can have complex calibration systems for settingthe voltage slope and offset of a glass pH probe. In an embodiment, avoltage sync is provided that resets a third-party pH meter. Withreference to FIG. 27A, the electronics unit includes three buttons topermit the electronics unit to emulate the three point calibration forcertain probe systems, which is standard prior to data collection withan analog pH meter. The electronics unit of FIG. 27A includes threebuttons, each button configured to enable the electronic unit to outputto an electronic system (for use with a traditional prove) a voltagevalue that is recognizable by the electronic system. For example, thebutton marked pH 7 (middle button) can sets the voltage output of thetransmitter to about zero volts. The button marked pH 4 (left button)can set the voltage to about +0.177 V (or any other voltagecorresponding to pH 4 for the traditional electronics system). Thebutton marked pH 10 (right button) can set the voltage to about −0.177 V(or any other voltage corresponding to pH 10 for the traditionalelectronics system). This way a pH meter that was calibrated to aparticular glass pH probe can be returned to the ideal slope and offset.

With continued reference to FIG. 27A, the electronic unit includes onecable or corrector for interfacing with electrochemical sensors providedherein (left cable), another connector for interfacing with atraditional electronics unit (right-top cable), such as an electronicsunit configured for use with glass probe electrochemical sensors (e.g.,pH sensors), and another connector (right-bottom cable) for providingpower to the electrochemical sensor.

While in certain embodiments a stand-alone electronics units isprovided, in other embodiments, the electronics units can be includedon-board the printed circuit board having the electrochemical sensor(see FIGS. 23 and 24).

FIG. 27B illustrates the electronic unit of FIG. 27A attached to (orelectrical communication with) a probe, in accordance with an embodimentof the invention. The probe can be any probe described herein, such as,for example, the probe of FIG. 1B. The probe comprises a cylindricalbody and a head portion. The head portion can include one or moreworking electrodes, a reference electrode and a counter electrode, asdescribed herein. The head portion is configured to come in contact witha solution having one or more analytes of interest, such as, e.g.,hydrogen ions (H+).

FIG. 27C shows a system having a probe attached to an electronics unit,such as the electronics unit of FIG. 27A, in accordance with anembodiment of the invention. The system further includes a reader (ormeter) for providing sensor measurements, such as pH measurements. In anembodiment, the reader is a digital reader. In another embodiment, theelectronic unit can be configured for enabling communication between areader, such as a third-party reader, and the probe. The third-partyreader can be configured for use with glass probe electrochemicalsensors, such as glass probe pH sensors.

In an embodiment, the electronics unit can be configured for wirelesscommunication with a probe. In another embodiment, the electronics unitcan include one or more of a WiFi transmitter, a Bluetooth transmitter,a radio frequency transmitter and an infrared (IR) transmitter forcommunicating with the probe.

Electronics Components for Communicating with External Devices

Another aspect of the invention provides an electrochemical sensor, suchas an electrochemical sensor on a printed circuit board, which includesa transmitter for wirelessly transmitting information to a systemconfigured to collect information from the electrochemical sensor. Thetransmitter can be reduced in size so that it can be included in thesensor (or probe). The transmitter can be configured to interface with areceiver on a system configured to communicate with the electrochemicalsensor. In an embodiment, the electrochemical sensor can be configuredfor WiFi transmission. In another embodiment, the electrochemical sensorcan be configured for Bluetooth transmission. In another embodiment, theelectrochemical sensor can be configured for radiofrequency (RF)transmission. In another embodiment, the electrochemical sensor can beconfigured for infrared transmission. In another embodiment, theelectrochemical sensor can be configured for inductive transmission(i.e., inductively coupling). In another embodiment, the electrochemicalsensor can be configured for optical (e.g., fiber optic) transmission.

In some embodiments, a transmitter is provided that is powered by anenergy storage device, such as a battery (e.g., lithium ion battery) ora photovoltaic (solar) cell. The energy storage device can be configuredto provide power to the transmitter over the life of the electrochemicalsensor. In an embodiment, the energy storage device is configured toprovide power to both the transmitter and the electrochemical sensor,such as any electrochemical sensor provided herein. In anotherembodiment, the transmitter can be an on-board transmitter. For example,the electrochemical sensor of FIG. 24, configured to be mounted at a tipof a probe assembly or shaft, can include a transmitter. The transmittercan include electronics that can provide a wireless link, such as adigital link (e.g., WiFi, Bluetooth) to a computer system. This link canbe done with WiFi, Bluetooth or other radio communications protocols,such as radiofrequency (RF) protocols.

An electrochemical sensor configured for wireless transmission can beused in numerous contexts. In an embodiment, an electrochemical sensorconfigured for wireless transmission can be configure to transmit sensordata from remote and hard-to-reach locations, such as providing datafrom inside oil wells, the human body (e.g., providing theconcentrations of various analytes, such as pH) and high pressuresettings, such as chemical reactors.

In another embodiment, an electronics component is provided that can beconfigured to provide a digital signal that can be read using acomputing device, such as a personal computer (PC) or a mobileelectronics device, such as an Apple® iPad® or iPod®, anAndroid®-enabled device, a Smart Phone, a netbook, a laptop, a tabletPC, or a slate PC.

In an embodiment, a graphical user interface (“GUI”) can provide a usera sensor reading, such as at a fixed point in time or as a function oftime. In an embodiment, the GUI can provide a user a sensor reading(e.g., pH reading) at least every 0.1 seconds, or 0.2 seconds, or 0.3seconds, or 0.4 seconds, or 0.5 seconds, or 0.6 seconds, or 0.7 seconds,or 0.8 seconds, or 0.9 seconds, or 1 second, or 1.1 seconds, or 1.2seconds, or 1.3 seconds, or 1.4 seconds, or 1.5 seconds, or 1.6 seconds,or 1.7 seconds, or 1.8 seconds, or 1.9 seconds, or 2 seconds, or 2.1seconds, or 2.2 seconds, or 2.3 seconds, or 2.4 seconds, or 2.5 seconds,or 2.6 seconds, or 2.7 seconds, or 2.8 seconds, or 2.9 seconds, or 3seconds, or 3.1 seconds, or 3.2 seconds, or 3.3 seconds, or 3.4 seconds,or 3.5 seconds, or 3.6 seconds, or 3.7 seconds, or 3.8 seconds, or 3.9seconds, or 4 seconds, or 4.1 seconds, or 4.2 seconds, or 4.3 seconds,or 4.4 seconds, or 4.5 seconds, or 4.6 seconds, or 4.7 seconds, or 4.8seconds, or 4.9 seconds, or 5 seconds, or 5.1 seconds, or 5.2 seconds,or 5.3 seconds, or 5.4 seconds, or 5.5 seconds, or 5.6 seconds, or 5.7seconds, or 5.8 seconds, or 5.9 seconds, or 6 seconds, or 6.1 seconds,or 6.2 seconds, or 6.3 seconds, or 6.4 seconds, or 6.5 seconds, or 6.6seconds, or 6.7 seconds, or 6.8 seconds, or 6.9 seconds, or 7 seconds,or 7.1 seconds, or 7.2 seconds, or 7.3 seconds, or 7.4 seconds, or 7.5seconds, or 7.6 seconds, or 7.7 seconds, or 7.8 seconds, or 7.9 seconds,or 8 seconds, or 8.1 seconds, or 8.2 seconds, or 8.3 seconds, or 8.4seconds, or 8.5 seconds, or 8.6 seconds, or 8.7 seconds, or 8.8 seconds,or 8.9 seconds, or 9 seconds, or 9.1 seconds, or 9.2 seconds, or 9.3seconds, or 9.4 seconds, or 9.5 seconds, or 9.6 seconds, or 9.7 seconds,or 9.8 seconds, or 9.9 seconds, or 10 seconds, or 10.1 seconds, or 10.2seconds, or 10.3 seconds, or 10.4 seconds, or 10.5 seconds, or 10.6seconds, or 10.7 seconds, or 10.8 seconds, or 10.9 seconds, or 11seconds, or 11.1 seconds, or 11.2 seconds, or 11.3 seconds, or 11.4seconds, or 11.5 seconds, or 11.6 seconds, or 11.7 seconds, or 11.8seconds, or 11.9 seconds, or 12 seconds, or 12.1 seconds, or 12.2seconds, or 12.3 seconds, or 12.4 seconds, or 12.5 seconds, or 12.6seconds, or 12.7 seconds, or 12.8 seconds, or 12.9 seconds, or 13seconds, or 13.1 seconds, or 13.2 seconds, or 13.3 seconds, or 13.4seconds, or 13.5 seconds, or 13.6 seconds, or 13.7 seconds, or 13.8seconds, or 13.9 seconds, or 14 seconds, or 14.1 seconds, or 14.2seconds, or 14.3 seconds, or 14.4 seconds, or 14.5 seconds, or 14.6seconds, or 14.7 seconds, or 14.8 seconds, or 14.9 seconds, or 15seconds, or 15.1 seconds, or 15.2 seconds, or 15.3 seconds, or 15.4seconds, or 15.5 seconds, or 15.6 seconds, or 15.7 seconds, or 15.8seconds, or 15.9 seconds, or 16 seconds, or 16.1 seconds, or 16.2seconds, or 16.3 seconds, or 16.4 seconds, or 16.5 seconds, or 16.6seconds, or 16.7 seconds, or 16.8 seconds, or 16.9 seconds, or 17seconds, or 17.1 seconds, or 17.2 seconds, or 17.3 seconds, or 17.4seconds, or 17.5 seconds, or 17.6 seconds, or 17.7 seconds, or 17.8seconds, or 17.9 seconds, or 18 seconds, or 18.1 seconds, or 18.2seconds, or 18.3 seconds, or 18.4 seconds, or 18.5 seconds, or 18.6seconds, or 18.7 seconds, or 18.8 seconds, or 18.9 seconds, or 19seconds, or 19.1 seconds, or 19.2 seconds, or 19.3 seconds, or 19.4seconds, or 19.5 seconds, or 19.6 seconds, or 19.7 seconds, or 19.8seconds, or 19.9 seconds, or 20 seconds or 20.1 seconds, or 20.2seconds, or 20.3 seconds, or 20.4 seconds, or 20.5 seconds, or 20.6seconds, or 20.7 seconds, or 20.8 seconds, or 20.9 seconds, or 21seconds, or 21.1 seconds, or 21.2 seconds, or 21.3 seconds, or 21.4seconds, or 21.5 seconds, or 21.6 seconds, or 21.7 seconds, or 21.8seconds, or 21.9 seconds, or 22 seconds, or 22.1 seconds, or 22.2seconds, or 22.3 seconds, or 22.4 seconds, or 22.5 seconds, or 22.6seconds, or 22.7 seconds, or 22.8 seconds, or 22.9 seconds, or 23seconds, or 23.1 seconds, or 23.2 seconds, or 23.3 seconds, or 23.4seconds, or 23.5 seconds, or 23.6 seconds, or 23.7 seconds, or 23.8seconds, or 23.9 seconds, or 24 seconds, or 24.1 seconds, or 24.2seconds, or 24.3 seconds, or 24.4 seconds, or 24.5 seconds, or 24.6seconds, or 24.7 seconds, or 24.8 seconds, or 24.9 seconds, or 25seconds, or 25.1 seconds, or 25.2 seconds, or 25.3 seconds, or 25.4seconds, or 25.5 seconds, or 25.6 seconds, or 25.7 seconds, or 25.8seconds, or 25.9 seconds, or 26 seconds, or 26.1 seconds, or 26.2seconds, or 26.3 seconds, or 26.4 seconds, or 26.5 seconds, or 26.6seconds, or 26.7 seconds, or 26.8 seconds, or 26.9 seconds, or 27seconds, or 27.1 seconds, or 27.2 seconds, or 27.3 seconds, or 27.4seconds, or 27.5 seconds, or 27.6 seconds, or 27.7 seconds, or 27.8seconds, or 27.9 seconds, or 28 seconds, or 28.1 seconds, or 28.2seconds, or 28.3 seconds, or 28.4 seconds, or 28.5 seconds, or 28.6seconds, or 28.7 seconds, or 28.8 seconds, or 28.9 seconds, or 29seconds, or 29.1 seconds, or 29.2 seconds, or 29.3 seconds, or 29.4seconds, or 29.5 seconds, or 29.6 seconds, or 29.7 seconds, or 29.8seconds, or 29.9 seconds, or 30 seconds, or 31 seconds, or 32 seconds,or 33 seconds, or 34 seconds, or 35 seconds, or 36 seconds, or 37seconds, or 38 seconds, or 38 seconds, or 40 seconds, or 45 seconds, or50 seconds, or 55 seconds, or 1 minute, or 2 minutes, or 3 minutes, or 4minutes, or 5 minutes, or 6 minutes, or 7 minutes, or 8 minutes, or 9minutes, or 10 minutes, or 20 minutes, or 30 minutes, or 1 hour, or 2hours, or 3 hours, or 4 hours, or 5 hours, or 6 hours, or 7 hours, or 8hours, or 9 hours, or 10 hours, or 11 hours, or 12 hours, or 1 day, or 2days, or 3 days, or 4 days, or 5 days.

In an embodiment, the electrochemical sensor can provide a reading to acomputer system coupled to (or interfaced with) the electrochemicalsensor. Such a reading can be recorded in the computer's memory with atimestamp that can be assembled into a date and/or time based graph. TheGUI can also record multiple sensors outputs, such as from a probehaving a plurality of electrochemical sensors (see FIGS. 22A and 22B).The GUI can compile data from different sensors and provide a user anoutput that is calculated from the multiple sensors. In an embodiment,such output can be an average output, such as an average pH. Forexample, if pH measurements are being made, the curve for pH can betemperature dependent, and the system can correct the pH fortemperature. The system can calculate and provide an input for thetemperature and concentration of hydrogen ions.

In an embodiment, a sensor is provided having electrochemical sensorsand sensors for measuring the temperature of a solution or fluid in thesolution or fluid. The temperature can be the temperatures as measuredin the vicinity of the sensor. In an embodiment, the sensor includes oneor more thermocouples for measuring the temperature of a solutionadjacent to the sensor. Such temperature measurement can be used toprovide a temperature-corrected sensor output, such as a pH outputcorrected for temperature.

In some embodiments, a digital meter is provided that can display thereadout from an electrochemical sensor. In an embodiment, the digitalmeter can provide real-time data. In another embodiment, the digitalmeter can provide date at set intervals. In such a case, data can beaveraged over a certain time period that can be a function of theintervals in which data is measured and/or displayed. The digital metercan provide one or more of current, voltage, impedance, conductivity,temperature, time, time left in the lifetime of the device (e.g., if theelectrochemical sensor is a time-limited device, see above), areacalculations (e.g., IV area calculations) and maximum peak positions inan IV curve. A sensor trend can also be displayed. In an embodiment, themeter can communicate digitally with industrial digital control units,such as control units in coal fired power plants, steam boiler water,food and beverage manufacturing facilities, personal care products,water and wastewater, refineries, biofuel manufacturing facilities,reactors, bioreactors, oil well rigs, oil well boars, or nuclear powerplants and the manufacture of radionucleide therapeutics.

Sensors for Insertion into Containers for Use with Glass Probes

Another aspect of the invention provides a sensor system comprising aredox-active moiety-containing analyte sensor for insertion into acontainer for use with a glass probe analyte sensor. In an embodiment,the redox-active moiety-containing analyte sensor comprises one or moreredox-active moieties. In another embodiment, the redox-activemoiety-containing analyte sensor comprises a redox-active moiety that issensitive to the presence of an analyte and another redox-active moietythat is insensitive to the presence of the analyte. In anotherembodiment, the redox-active moiety-containing analyte sensor isdisposed in a probe body having a form factor configured for insertioninto a container for use with a glass probe analyte sensor.

In an embodiment, an electrochemical sensor is provided having a sensorhead, the sensor head having working, counter and reference electrodes,as described above. The sensor head can be mounted to a probe bodyhaving a shape (size and length) configured to mate with existing ortraditional probe systems, such as glass probe systems. Theelectrochemical sensor can be interfaced with a system used to operatethe traditional probe system with the aid of the electrical boxdescribed above in the context of FIG. 27.

With reference to FIG. 28, an electrochemical probe and transmitter areillustrated for use with a bioreactor, in accordance with an embodimentof the invention. The electrochemical probe is connected to anelectronics component that can be used to interface the electrochemicalprobe with an existing (or third-party) system or device configured tocommunicate with (or take measurements from) a probe, such as, forexample, a glass probe. The pH probe includes a probe head at a distalend of a probe body. The probe body is configured to extend into thebioreactor for taking measurements of analytes of interests (e.g., H+for pH measurements).

In some embodiments, a sensor is provided for insertion into a containerconfigured for use with a traditional or conventional glass probe. In anembodiment, the container is cylindrical in shape. In anotherembodiment, the container has a circular cross-section. In anotherembodiment, the container is formed of one or more metals. In anotherembodiment, the container is formed of one or more metals includingaluminum, nickel, platinum, ruthenium, rhodium, tungsten, titanium,palladium, copper, silver, gold and iron. In another embodiment, thecontainer is formed of one or more metals including stainless steel.

In an embodiment, an electrochemical sensor is provided having a headportion comprising one or more working electrodes, a counter electrodeand a reference electrode, the head portion mounted on a body portion,such as a tubular or cylindrical body portion. The body portion caninclude threading configured to permit the body portion to be mounted toa bioreactor. In an embodiment, the threading includes PG-13 threading.In an embodiment, the body portion can be formed of a polymericmaterial. In another embodiment, the body portion can be formed of ametallic material, such as stainless steel.

Calibration

Another aspect of the invention provides a system for calibrating anelectrochemical sensor with the aid of a robot. In an embodiment, arobot is provided to obtain calibration parameters, which cansubsequently be provided to uses or stored in an EEPROM onboard anelectrochemical sensor.

In an embodiment, a robot comprises a robot arm for placing anelectrochemical sensor in a first solution or environment having a knownanalyte, such as a solution or environment having a known pH. The robotthen measures the output of the electrochemical sensor for theparticular analyte measurements and records the output, such as in atable. The robot then places the electrochemical in a second solution orenvironment having a known analyte, such as a solution or environmenthaving a known pH. The robot then measures the output of theelectrochemical sensor for the particular analyte measurement andrecords the output, such as in a table. The robot can repeat thisprocess, as desired, until a predetermined number of measurements havebeen made. For example, the robot can record electrochemical sensoroutputs for pH 4, pH 7 and pH 10 solutions. The robot can then recordthe calibration data in a calibration file for distribution to a user,or record the calibration data in a memory chip, such as a memory chiponboard the electrochemical sensor (e.g., EEPROM).

The pH sensors of the present invention are amenable to miniaturization.In an embodiment, electrochemical sensors of the present invention, suchas pH sensors, can be miniaturized for use in capsules for insertion ina subject's body. In another embodiment, electrochemical sensors of thepresent invention, such as pH sensors, can be miniaturized for use incapsules for insertion in a subject's body. In another embodiment,electrochemical sensors of the present invention, such as pH sensors,can be miniaturized for use in skin patches applicable to a subject'sbody. In another embodiment, electrochemical sensors of the presentinvention, such as pH sensors, can be miniaturized for use a nucleicacid sequencing array (e.g., 512×512 array).

Methods and Systems for Determining pH from Voltammetric or AmperometricData

Another aspect of the invention provides systems and methods forconverting peaks in voltammetric or amperometric data to pH values. Insome embodiments, a system is provided for implementing the methodsprovided herein. Such methods can be implemented with the aid of asystem implementing an algorithm, as set forth in machine-readable code.

In an embodiment, in a first step, the system identifies all peakshaving at least 10 fold greater intensity than background noise level. Apeak is defined as a current maximum that is bordered by two currentminimums, one minimum on each side of the maximum. In an embodiment, amaximum can be 5% higher than a local minimum. The system then ranks themaxima in terms of height (value of the current at a given voltage),from maximum height to minimum height. Next, using the largest maximum(i.e., the maximum having largest value), the system takes the positionsof the local minima on each side of the largest maximum. The system thenfinds the equation of the line that connects the two local minima. In anembodiment, the line has the form of I=c+mV, wherein ‘I’ is the measuredcurrent, ‘V’ is the applied voltage, ‘m’ is the slope of the line and‘c’ is the intercept. This process is repeated for each data point inthe amperometric data. For a set of three data points a correctionequation is provided. The system then subtracts the correction equationfor each original data point, (I_(n), V_(n)). A corrected data point isthen achieved, represented by (I_(nc), V_(n))=(I_(n)−(c+m*V_(n)),V_(n)). This correction removes other phenomena from the data, such aslocal abnormalities due to noise.

Next, with a corrected data set, the system finds the voltage of amaximum current of interest. In an embodiment, a search for the voltageis limited to data points between two local minima, as described above.The system first finds the difference between a maximum and the minima.The system then takes the difference between the higher minima and themaximum current. In an embodiment, only the data that is above apredetermined percentage of the current range of interest is used. In anembodiment, only data that is above a 50%, or 55%, or 60%, or 65%, or70%, or 75%, or 80%, or 85%, or 90% of the current range of interest isused. In another embodiment, only data that is from about 20% to 100% ofthe current range of interest is used. For example, the upper 80% ofdata between the higher minima and the maximum can be used. Next, thedata thus generated is fit to a parabola. While a parabola can becomputationally desirable, other mathematical functions can be used tofit the data. For example, the data can be fit with a Taylor Seriesexpansion model to an order that minimizes the spread among the datapoints and points generated by the model. Next, the maximum of thefitted parabola (or other function) can be used as the maximum of theamperometric curve. The distance between the corrected maxima for thefirst working electrode (WE1) and the second working electrode (WE2) isused to determine a pH. In an embodiment, the pH can be represented bypH=c+m(WE1−WE2)+m2(WE1−WE2)²+m3(WE1−WE2)³, wherein ‘c’, ‘m’, ‘m2’ and‘m3’ are constants that are determined experimentally—i.e., theconstants are determined by fitting the equation to known one or moresamples having known pH values).

Form Factors and Sensor Applications

Another aspect of the invention provides electrochemical sensors havingform factors for use with various applications, such as insertion into acontainer for use with glass probe sensors. This advantageously enablesa user to replace glass probe sensor with electrochemical sensorsprovided herein, such as redox-active moiety containing sensors.

Sensors provided herein can be suited for various applications. In someembodiments, sensors provided herein are configured for use inbioreactors, such as, e.g., single-use and/or disposable bioreactors. Inother embodiments, sensors provided herein are configured for use insample preparation and/or analytical systems, such as chromatography,including, for example, liquid chromatography (e.g., high pressureliquid chromatography), gas chromatography, affinity chromatography,supercritical fluid chromatography, ion exchange chromatography,size-exclusion chromatography, reversed phase chromatography,two-dimensional chromatography, simulated moving-bed chromatography,pyrolysis gas chromatography, fast protein liquid chromatography,countercurrent chromatography, chiral chromatography, and columnchromatography. In other embodiments, sensors provided herein areconfigured for use in process separation unit operations, such asdistillation columns and absorption columns. In other embodiments,sensors provided herein are configured for use in continuous stirredtank reactors. In other embodiments, sensors provided herein areconfigured for use in plug flow rectors. In other embodiments, sensorsprovided herein are configured for use in fluidized bed reactors.

Sensors can have form factors (e.g., shapes, sizes) suited for suchvarious sensor applications provided herein. FIGS. 32A-32E showexemplary sensors having form factors suited for various applications.FIG. 32A shows a sensor having a form factor as found in someconventional probes. A redox-active moiety containing sensor 3201 isdisposed at a probe tip of the sensor of FIG. 32A. FIG. 32B shows anelectrochemical sensor having a form factor that is suited for use in pHsensors. The sensor of FIG. 32B can be used at the probe tip of thesensor of FIG. 32A. FIG. 32C shows a substantially flat sensor. Thesensor of FIG. 32C can be suited for use in reactors, such asbioreactors. The sensor of FIG. 32C can have a distribution of workingelectrodes as described herein. FIG. 32D shows a sensor configured foruse with in-line flow systems, such as, for example, plug flow reactors.A redox-active moiety containing sensor 3202 is disposed at a probe tipof the sensor of FIG. 32D. FIG. 32E shows a sensor configured for use ina system having a predetermined volume, such as, e.g., about 30microliters of retained volume.

Sensors provided herein can have supports for enabling the sensors to beused in various applications. For example, the sensors of FIGS. 32A, 32Dand 32E can have threads, o-rings, and hex bodies that allow for thesensors to be mounted in various settings, such as, for example,reactors (e.g., bioreactors), various unit operations (e.g.,distillation columns), flow-through systems and fermentors.

EXAMPLES Reagents and Instrumentations

Vinylferrocene, vinylanthracene, hydrofluoric acid were purchased fromSigma-Aldrich (Sigma-Aldrich INC., USA), ferrocenecarboxaldehyde andmesitylene were obtained from Alfa Aesar (Alfa Aesar INC., USA), and9-anthracene-carboxaldehyde was obtained from Acros Organics (AcrosOrganics INC. USA). All the chemicals were obtained with the highestgrade available and were used without further purification.

Different single-side polished, primary flat, 500 μm thick siliconwafers with (111) and (100) orientation were purchased from VirginiaSemiconductor with the following specification: i) P-type (100, 10-90Ω-cm resistivity), ii) P-type (100, 0.001-0.005 Ω-cm resistivity), iii)N-type (100, 10-40 Ω-cm resistivity), iv) N-type (100, 0.02-0.05 Ω-cmresistivity), v) P-type (111, 0.001-0.004 Ω-cm resistivity) and vi)N-type (111, 0.001-0.005 Ω-cm resistivity).

Electrochemical measurements were recorded using an Autolab computercontrolled potentiostat (Ecochemie, Utrecht, Netherlands) with astandard three-electrode configuration, consisting of a saturatedcalomel reference electrode (SCE, Radiometer, Copenhagen, Denmark), aplatinum auxiliary electrode (Bioanalytical Systems INC., USA)) andsilicon (Virginia Semiconductor INC, USA) working electrode.

Different pH solutions in the range of 1 to 12 were also prepared indeionized water as follows: pH 1.2, 0.10 M perchloric acid; pH 2.2, 0.05M perchloric acid; pH 4.6, 0.1 M acetic acid+0.10 M sodium acetate; pH5.6, 0.5 M sodium acetate; pH 6.5, 0.025 M K₂PO₄+0.025 M KH₂PO₄; pH7.33, 0.05 M K₂PO₄; pH 9.3, 0.10 M sodium borate; pH 13.5, 0.1 M sodiumhydroxide. These solutions also contained an addition of 0.10 M sodiumperchlorate as supporting electrolyte. The pH of these solutions wasmeasured using the SevinMulti (Mettler Toledo) pH meter.

Example 1 Preparation of H-terminated silicon surface

Silicon wafers (oriented (111) or (100), cut into ca. 1×1 cm² pieces)were cleaned using “Piranha” solution (concentrated H₂SO₄:30% H₂O₂, 3:1,v/v) at about 80° C. for 30 min and rinsed thoroughly with deionizedwater. (In some cases, smaller pieces, e.g., 2 mm×3.3 mm or 2 mm×2.7 mmwere used.) Subsequently, the wafer pieces were oxidized in H₂O₂:HCl:H₂O(2:1:8) at about 80° C. for 15 min, and in H₂O₂:NH₄OH:H₂O (2:1:8) atabout 80° C. for another 15 min, rinsed copiously with deionized water.The cleaned Si(100) wafers pieces were then etched in 2.5% HF solutionfor about 15 min. These procedures eliminate the native silicon oxidelayer, yielding an H-terminated surface. The H-terminated substrateswere quickly rinsed with deionized water, dried with nitrogen gas andwere used immediately for the derivatization experiments. The Si(100)(10-90 Ω-cm, P-type) was used for the experiments below.

Example 2 Derivatization of H-terminated silicon surface with ferrocenemoieties

Approximately 10 mmol mesitylene solution of vinylferrocene (VFc) orferrocenecarboxaldehyde (FcA) was put in a round bottom flask andbubbled with nitrogen or argon gas for at least 30 min. A piece of theH-terminated silicon substrate was then immersed in the solution andallowed to react with VFc or FcA for about 12 h under reflux at about150° C. in an oil bath. During the reaction, the solution was alsopurged with nitrogen (or argon) to eliminate dissolved oxygen and toprevent the substrate from being oxidized. After the reaction, thesubstrate derivatized with VFc or FcA was rinsed with dichloromethane,acetonitrile, and methanol; and dried under a stream of nitrogen gas.The derivatization of the H-terminated surface with ferrocene moietiesas described in Example 2 is illustrated in FIG. 5.

Example 3 Derivatization of H-Terminated Silicon Surface with AnthraceneMoieties

Approximately 10 mmol mesitylene solution of vinylanthracene (VA) oranthraldehyde (AnA) was put in a round bottom flask and bubbled withnitrogen or argon gas for at least 30 min. A piece of the H-terminatedsilicon substrate was then immersed in the solution and allowed to reactwith VA or AnA for about 12 h under reflux at about 150° C. in an oilbath. During the reaction, the solution was also purged with nitrogen(or argon) to eliminate dissolved oxygen and to prevent the substratefrom being oxidized. After the reaction, the substrate derivatized withVA or AnA was rinsed with dichloromethane, acetonitrile and methanol;and dried under a stream of nitrogen gas. The derivatization of theH-terminated surface with anthracene moieties as described in Example 3is illustrated in FIG. 6.

Example 4 Derivatization of H-Terminated Silicon Surface with Both theAnthracene and Ferrocene Moieties

A 10 mmol mesitylene solution of anthracene (VA or AnA) and ferrocene(VFc or FcA) in 1:1 ratio was put in a round bottom flask and bubbledwith nitrogen or argon gas for at least 30 min. A piece of theH-terminated silicon substrate was then immersed in the solution andallowed to react with the anthracene and ferrocene mixtures for about 12hours under reflux at 150° C. in oil bath. During the reaction, thesolution was also purged with nitrogen (or argon) to eliminate dissolvedoxygen and to prevent the substrate from being oxidized. After thereaction, the derivatized substrate was rinsed with dichloromethane,acetonitrile and methanol, and dried under a stream of nitrogen gas.FIG. 7 illustrates a reaction in which the silicon surface derivatizedwith both the anthracene and ferrocene using VFc and VA.

Example 5 Electrochemical Measurements of the Derivatized Silicon Wafersin Different pH Solutions

Square wave voltammetry (SWV) was carried out for the derivatizedsilicon wafers in a specially designed electrochemical cell as shown inFIG. 8. The electrochemical measurements were performed using a standardthree-electrode configuration. In these experiments, the derivatizedsilicon wafers were used as the working electrode, and was exposed todifferent pH solutions (about 10 mL) in the electrochemical cell. SWVwere performed with a frequency of 10 Hz, a step potential of 2 mV andan amplitude of 25 mV.

The amperometric response of the anthracene or ferrocene derivatizedsilicon substrate at different pH solutions was studied using SWV. SWVwas used as the electrochemical probe of the system because it producesa well-defined voltammetric peak in a single sweep. The correspondingsquare wave voltammograms recorded using a derivatized ferroceneelectrode at different pH solutions from pH 1.23 to 9.33 are shown inFIG. 9. These voltammograms show that as the pH values increase, thepeak potential of the ferrocene peaks remain at the same peak potential.These results show that ferrocene is a pH insensitive molecule which canact as an internal reference material.

The corresponding SW voltammograms recorded using a derivatizedanthracene electrode at different pH solutions, from pH 1.23 to 13.63,are shown in FIG. 10 (a). These voltammograms show that as the pH valueincreases, the peak potential attributed to the anthracene shifts to amore negative potential. The corresponding plot of the peak potentialagainst different pH is given in FIG. 10( b). The plot reveals a linearresponse from pH 1 to pH 14 with a corresponding gradient of ca 55.1 mVper pH unit. The ability of anthracene compounds to act as pH sensitivemolecule is thus demonstrated.

The corresponding SW voltammograms recorded using a derivatizedferrocene+anthracene electrode at different pH solutions, from pH 1.23to 9.33, are shown in FIG. 11 (a). These voltammograms illustrate thatas the pH values increase, the ferrocene peak remains at the same peakpotential while the anthracene peak shifts to a more negative potential.The corresponding plot of the difference between the two peak potentialsversus pH is shown in FIG. 11( b). The plot reveals a linear responsefrom pH 1 to pH 9.33 with a corresponding gradient of ca 45.1 mV per pHunit.

Example 6 Heat Stability

A SW voltammogram was recorded for the silicon wafer derivatized withboth VA and FcA moieties at room temperature in pH 6.52 buffer. Thederivatized silicon sample was then autoclaved in a Consolidated Stills& Sterilizers for 40 min, and a SW voltammogram was recorded in pH 6.52buffer after the autoclave. Next, the same sample was autoclaved againunder the same condition 10 times, with a SW voltammogram recorded in pH6.52 buffer after each autoclave.

The heat stability testing of the pH sensor was performed using a FcA+VAderivatized silicon sample. The resultant voltammograms are shown inFIG. 12. A decrease in both the ferrocene and anthracene currents wereobserved after the first autoclave. Thereafter, both the peak currentsremain relatively stable though subsequent cycles of autoclaving; infact the peaks remain stable for ten cycles, showing that the sensor canwithstand repeated heat sterilization.

Example 7 Fouling Test

Four SW voltammograms were recorded for the four separate silicon wafersderivatized with FcA and AnA moieties at room temperature in pH 6.52buffer. These derivatized samples were then autoclaved in theConsolidated Stills & Sterilizers for 20 min and were immersed in a 5 mLcell culture fermentation medium for six days. Then these samples weretaken out of the cell culture medium, and SW voltammograms were recordedin pH 6.52 buffer again. SW voltammetry was also carried out in the cellculture medium, using the same silicon wafers.

The fouling testing of the pH sensor was performed using fivederivatized silicon samples. The resultant voltammograms were shown inFIG. 13. In all cases, the anthracene peaks remain stable with after sixdays exposure. The ferrocene peaks decrease after exposure, but theferrocene peaks are still identifiable. These findings demonstrate thatthe pH sensors are still in good working order after six days exposureto the cell culture environment, demonstrating that the ability of thederivatized sensor to resist fouling. A control sensor was incubated for6 days in culture fluid without cells or secreted proteins. Thissensor's voltammogram exhibited a similar profile to the four that hadbeen incubated in the actual cell fermentation environment, (FIG. 14)suggesting that any loss of signal amplitude was not a function ofcellular debris deposition.

Example 8 Stability of the Anthracene- and Ferrocene-Derivatized SiSurfaces

The stability testing of the Ac+Fc derivatized silicon surface wasconducted for 22 days under continuous electrochemical measurement usingPGSTAT12 autolab potentiostat in pH 4.65 buffer medium, scanning from−0.8 V to 0.8 V vs. Ag. The Ac peak remained as a well-defined peak witha full width half maximum (FWHM) of 60 mV throughout the course of theexperiments, while the Fc peaks broadened with time. Although the Fcpeaks became broader, they are still identifiable and can be used as areference. These findings demonstrate that the two-component derivatizedsilicon surface are still in good working order after 22 days continuousoperation in pH 4.65 buffer medium, demonstrating the long termstability of the derivatized surface in the buffer medium.

In general, the Fc peaks are most well-defined and stable when the Fcmolecules were derivatized onto a heavily doped silicon substrate. FIG.15( a) depicts SW voltammetric responses FcA on Si(100, N-type, 1-5 mΩcm) in pH 7.33 buffer medium, showing every 50^(th) scan of the 2,500consecutive runs.; FIG. 15( b) depicts voltammetric responses of VFc onSi(111, N-type, 0.02-0.05 cm in pH 7.33 buffer medium, showing every50^(th) scan of the 2,500 consecutive runs. In some cases, both VFc andFcA moieties behave better on an N-type substrate than a P-typesubstrate in terms of the size of the peak current produced. The Fcderivatized silicon surfaces were all pH insensitive, i.e., the Fc peakdoes not shift upon exposure to different pH environments.

Example 9 Temperature Variation

The Nernst equation provides a theoretical framework for evaluating thetemperature dependence of redox active species. It predicts that theslope of the peak potential against pH plot will increase as thetemperature increases:

$\begin{matrix}{E_{p} = {E_{f}^{0} - {\frac{2.3{RTm}}{nF}{pH}}}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

Where E_(p), is the peak potential (V), E_(f) ⁰ is the standardelectrode potential (V), R is the universal gas constant (J K⁻¹ mol⁻¹),T is the absolute temperature (K), F is the Faraday constant (C mol⁻¹),m and n are the number of protons and electrons involved in the redoxreaction, respectively. In the case of anthracene, the redox process forsuch molecules in the aqueous solution involves the participation of 2electrons and 2 protons, thus m=n=2.

SW voltammograms were recorded for three pH solutions at pH 4.65, 7.33and 9.35. FIG. 16 shows the overlaid SW voltammograms of Ac derivatizedsilicon over the temperature range of 8 to 56° C. in pH 7.33 buffermedium. Similar responses were obtained at pH 4.65 and pH 9.35. There isa shift of the peak potential to more negative values with increasingtemperature which may be attributed in part to a combination of changesin the reference couple, the temperature dependence of the formalpotential (E_(f) ⁰), and the temperature term in Eq. 1. Analysis of theslope of the peak potential as a function of pH obtained experimentallyat each temperature is tabulated in Table 1, which illustrates that theslope of pH against peak potential plot varies with temperature. Thetheoretical slope as predicted by the Nernst equation as seen in Eq. 1is also listed in Table 1 for comparison. As can be seen, the variationof the gradient of peak potential with pH is not Nernstian and indeed isrelatively insensitive of temperature varying by about 2 mV/pH unit overa temperature range of ˜50° C. This dependence can be compared to a 10mV/pH shift that can be observed for a glass electrode. This small shiftwith temperature is beneficial in that it not only demonstrates thatthese Ac derivatized silicon wafers may be used as pH sensors atelevated temperatures, but also that they are not greatly affected bychanges in temperature.

TABLE 1 A comparison of the theoretically predicted slope andexperimentally obtained slope of the plot of pH against Ac peakpotential for a range of temperatures. T (° C.) 8 17 28 44 56 T (K) 281290 301 317 329 Theoretical (mV/pH) 55.7 57.5 59.7 62.8 65.2Experimental (mV/pH) 55.0 55.5 56 56.3 57.0

Example 10 Stability During Active Measurement in Cell Culture Medium

A fouling testing was carried out using anthracene derivatized silicon(100) wafer in the incubator (under controlled temperature of 37° C. andCO₂ content of 5%) with continuous electrochemical measurements in cellculture (LP VA) medium for 7 days using a three-electrode setupconnected to an μautolab type III potentiostat. The three-electrodesetup (in an electrochemical cell) was autoclaved in a ConsolidatedStills & Sterilizers autoclave for 40 min prior to adding 5 mL of thecell culture medium to the autoclaved setup. The electrochemical cellcontaining the medium was then transferred to the incubator, wherecontinuous electrochemical measurements were performed.

Voltammograms were taken repeatedly over 7 days resulting in 10,000consecutive voltammograms. FIG. 18( a) shows voltammograms taken overthe 7 day period (every 250^(th) scan of the 10,000 consecutive runs).The anthracene peak was observed at ˜0.71V vs. Ag and remainedconsistent throughout the 7 days of in-situ electrochemical measurementsin the cell culture medium. A plot of the anthracene peak potential overthe 7 day time period is shown in FIG. 18( b). These findingsdemonstrate that the anthracene derivatized silicon is still in goodworking order and that the peak potential remains consistent while thesensor is actively sensing the solution for 7 days.

Example 11 Derivatization of H-Terminated Silicon Surfaces with2-Allyl-1-Hydroxy-Anthraquinone

Into a round bottom flask containing 50 mL of mesitylene was placed 5 mgof 2-allyl-1-hydroxy-anthraquinone. Nitrogen was bubbled for at least 30minutes. Subsequently, a H-terminated silicon wafer was placed into theflask and was allowed to react with 2-allyl-1-hydroxy-anthraquinone for12 hours under reflux at 150° C. in an oil bath. During the reaction,the solution was purged with nitrogen gas to prevent the substrate frombeing re-oxidized. Following the reaction, the2-allyl-1-hydroxy-anthraquinone derivatized silicon wafer was rinsedwith dichloromethane, hexane, then methanol and dried under a stream ofnitrogen.

Example 12 Derivatization of Silicon Wafers with Ferrocene Using aSemiconductor Oxide Surface

Si(100) wafer pieces were cleaned using “Piranha” solution (3:1 v/vconcentrated H2SO4/30% H2O2) for 30 minutes at 100° C. and rinsedthoroughly with deionized water. Subsequently, the wafers were oxidizedin 2:1:8 HCl/H2O2/H2O for 15 minutes at 80° C. followed by 2:1:8NH4OH/H2O₂/H2O for 15 minutes at 80° C. and then rinsed with copiousamounts of water. The oxidized silicon wafers were then immersed in 2%3-aminopropyltriethoxysilane (APTES) solution in acetone for 2 minutesfollowed by rinsing with acetone to form an amino-terminated monolayer.This was then allowed to react with 10 mg/mL dicyclohexyl-carbodiimide(DCC) and 50 mg/mL ferrocene carboxylic acid in DMSO for 12 hours.Following the reaction, the ferrocene derivatized silicon wafer wasrinsed with dichloromethane, acetone, and methanol and then dried undera stream of nitrogen.

Example 13 Derivatization of Silicon Wafers with Anthracene Using aSemiconductor Oxide Surface

Si(100) wafer pieces were cleaned using “Piranha” solution (3:1 v/vconcentrated H2SO4/30% H2O2) for 30 minutes at 100° C. and rinsedthoroughly with deionized water. Subsequently, the wafers were oxidizedin 2:1:8 HCl/H2O2/H2O for 15 minutes at 80° C. followed by 2:1:8NH4OH/H2O2/H2O for 15 minutes at 80° C. and then rinsed with copiousamounts of water. The oxidized silicon wafers were then immersed in 2%3-APTES solution in acetone for 2 minutes followed by rinsing withacetone to form an amino-terminated monolayer. This was then allowed toreact with 10 mg/mL dicyclohexyl-carbodiimide (DCC) and 50 mg/mLanthracene carboxylic acid in DMSO for 12 hours. Following the reaction,the anthracene derivatized silicon wafer was rinsed withdichloromethane, acetone and methanol and then dried under a stream ofnitrogen.

Example 14 Ferrocene Covalently Attached to Various Doped Si(100)Substrates

Ferrocene was covalently attached to the following four silicon surfacesvia an H-terminated silicon substrate and vinyl ferrocene: i) Si (100)N-type (0.02-0.05 ohm-cm)—highly-doped N-type, ii) Si (100) P-type(0.005-0.020 ohm-cm)—highly-doped P-type, iii) Si (100) N-type (10-40ohm-cm)—lightly-doped N-type, and iv) Si (100) P-type (10-90ohm-cm)—highly-doped P-type. A well-defined ferrocene peak was observedfor all four substrates. The highly-doped substrates in general produceda larger electrochemical current than the corresponding lightly-dopedsubstrates. While not being bound by theory, this difference may beexplained by the fact that the highly-doped substrates contain morecharge carriers, i.e., are more conductive. Similar observations wereobserved when the electrochemistry was performed in a solution offerrocene carboxaldehyde. FIG. 19 has charts showing the peak current ofsilicon substrates derivatized with (a) vinyl ferrocene and (b)ferrocene carboxaldehyde in pH 1.63 solution on the four types of dopedsilicon.

Example 15 Four Electrode System

A system was constructed having four electrodes comprising (i) a counterelectrode, (ii) a reference electrode, (iii) a highly-doped N-typesilicon wafer derivatized with vinyl ferrocene, and (iv) a lightly-dopedP-type silicon wafer derivatized with anthracene carboxaldehyde. FIG. 20illustrates the voltammograms obtained with the four electrode systemconsisting of a highly-doped N-type silicon wafer derivatized with vinylferrocene and a lightly-doped P-type silicon wafer derivatized withanthracene carboxaldehyde. The voltammograms were obtained in pH 7.03solution and the peak potentials were stable over 200 consecutive scans(every 50th scan is shown).

Example 16 Working Electrodes Coated with Nafion Membranes

A Nafion membrane was applied to a working electrode having ferrocene. A5 mil thick section of pre-formed Nafion membrane (N115) was pre-treatedeither by soaking in water or in an acid solution (about 5% HCl or 5MHNO₃) at about 100° C. for about 1 hr. The treated membrane was then cutto a size appropriate to cover the surface of the working electrode. TheNafion membrane was then coated with a liquid dispersion of Nafion 117or Nafion 2020, and applied directly to the electrode surface. Themembrane-coated electrode was then cured in a regular or vacuum oven atabout 120° C. for about 1 hr. The electrode was then rehydrated andtested. Testing showed stability as to the peak position in SWV.

Example 17 Nafion-Plastic Composites

A porous plastic membrane (pore size 75-100 micron, 0.5 mm thick) is cutinto a 1 cm disk. The disk is saturated with a Nafion dispersion andallowed to dry for 30 min at 50° C. After a second cycle of saturationand drying, the disk is placed in a 120° C. oven for about 1 hour. Asingle electrode is then wetted with the Nafion dispersion and placed onthe smooth side of the disk. This assembly is then cured at 120° C. forabout 1 hour. The backside of the electrode is then coated with aconductive epoxy and a wire is affixed. The assembly is potted into awaterproof fixture for electrochemical measurements.

Example 18 Light Sensitivity

A sensor has a first working electrode (WE1) and second workingelectrode (WE2) that are formed of silicon. WE1 includes a layer offerrocene and WE2 includes a layer of anthracene. A first probe has WE1and WE2 that are shielded from light with PES layers, and a second probehas WE1 and WE2 that are not shielded (or unshielded) from light. Thefirst probe and second probe are inserted into a solution having a pH ofabout 4. Continuous pH measurements are made and plotted as thedifference between the peak potentials of WE1 and WE2. Sensor outputs(y-axis, mV) as a function of time (x-axis) during pH measurements forboth the shielded (dashed line, top) and unshielded (solid line, bottom)probes are shown in FIG. 30. The output of the shielded sensor uponexposure to light is different than the output of the unshielded sensorupon exposure to light. During pH measurements, the output of theunshielded sensor shows a sensor response that is coincident withexposure to light; the output of the shielded sensor does not exhibitsuch a behavior. Sensor output when the first and second workingelectrodes are shielded (top) does not display the light-sensitivebehavior of the unshielded working electrodes (bottom). The figure alsoshows the effect of either infrared (IR) or room light for both shieldedand unshielded probes. The shielded sensor is unresponsive to IR or roomlight.

Example 19 The Effect of Light on Sensor Output

A sensor has a first working electrode (WE1) and second workingelectrode (WE2) that are formed of silicon. WE1 includes a layer offerrocene and WE2 includes a layer of anthracene. The sensor is immersedin various solutions, each solution having a predetermined pH. Sensoroutput is recorded both with and without exposure of the sensor tolight. FIGS. 31A-31F show sensor output (y-axis; current, arbitraryunits) at various pH's and under light and dark conditions as a functionof voltage (mV). Channel 2 corresponds to the sensor output for WE2.Also provided in the figures are Channel 2 peak positions (mV). FIG. 31Ashows the sensor output at pH 5 without exposure to light (i.e., thesensor is in the dark). FIG. 31B shows the sensor output at pH 5 whilethe sensor is exposed to light. FIG. 31C shows the sensor output at pH 7without exposure to light. FIG. 31D shows the sensor output at pH 7while the sensor is exposed to light. FIG. 31E shows the sensor outputat pH 10 without exposure to light. FIG. 31F shows the sensor output atpH 10 while the sensor is exposed to light. The Channel 2 signal(current) when the sensor is exposed to light is more intense thansituations in which pH measurements are made without exposure to light.This indicates that, when the sensor is exposed to light, better signalto noise may be achieved.

Example 20 The Effect of Light on Sensor Output

A sensor has a first working electrode (WE1) and second workingelectrode (WE2) that are formed of silicon. WE1 includes a layer offerrocene and WE2 includes a layer of anthracene. The sensor is immersedin a solution with a pH of 5. Sensor output is recorded both with andwithout exposure of the sensor (and WE1 and WE2) to light of fixedintensities and wavelengths. Light is exposed on sensor surfaces havingthe ferrocene and anthracene moieties. It is observed that peak shapesare sharper in the presence of light up to a certain intensity andwavelength. At higher intensities the signal is destroyed. Optimalresponses are obtained in the IR or near-IR wavelength range, such aslight having a wavelength greater than or equal to about 750 nm, orgreater than or equal to about 850 nm.

Example 21 Doping Configurations

Multiple sensors are formed from silicon wafers having varying dopingconfigurations. Each sensor has a first working electrode (WE1) andsecond working electrode (WE2) that are formed of silicon. WE1 includesa layer of ferrocene and WE2 includes a layer of anthracene. During pHmeasurements, electrochemical responses from anthracene-coveredelectrodes (WE2) are observed when anthracene is derivatized onto thefollowing silicon surfaces: Si(100) wafer, p-type, resistivity betweenabout 1 and 20 Ω-cm; Si(100) wafer, p-type, resistivity between about 1and 90 Ω-cm; Si(111) wafer, p-type, resistivity between about 1 and 20Ω-cm; and Si(111) wafer, p-type, resistivity between about 1 and 10Ω-cm. An anthracene signal with an appreciable signal-to-noise ratio isnot observed when derivatized on an n-type silicon substrate. Ananthracene signal is not observed when derivatized on a p-type siliconsubstrate with resistivity less than about 1 Ω-cm. Electrochemicalresponses are observed for ferrocene on both p-type and n-type silicon(both Si(100) and Si(111). Signals for Ferrocene on all resistivities ofp-type silicon and on n-type silicon with resistivities <5 μΩ-cm areunstable over time (in some situations, it is observed that the signaldegrades with continuous square wave voltammetric scanning). Ferroceneon n-type surfaces with resistivities between 1 and 90 Ω-cm are stable.

Example 22 Sensor Stability

A test was conducted to monitor fermentation in a fermentation reactor.The pH of the reactant (i.e., cell culture) and product content of thefermentation reactor was monitored over a 10-day period using both aredox-active moiety-based pH sensor (see above) and a conventional glasselectrode sensor (Applikon Biotechnology). Both sensors were autoclavedwithin the bioreactor prior to the initiation of the cell culture. Theredox-active moiety-based pH sensor was not recalibrated during the10-day period. FIG. 33 shows the pH of the contents of the fermentationreactor over the 10-day period. The redox-active moiety-based pH sensortracked the glass electrode sensor without any appreciable drift andwithout exhibiting any appreciable electronic background noisethroughout the course of the 10-day period. In contrast, the glasselectrode sensor exhibited electronic noise during the 10-day period.

Electrochemical sensor systems, devices and methods provided herein canbe combined with or modified by other systems, devices and methods. Forexample, electrochemical sensors described herein, including methods forforming such sensors, can be combined with or modified by systems andmethods described in U.S. patent application Ser. No. 12/049,230 to Kahnet al. (“SILICON ELECTROCHEMICAL SENSORS”) and PCT/US2008/066165 to Kahnet al. (“SEMICONDUCTOR ELECTROCHEMICAL SENSORS”), which applications areentirely incorporated herein by reference.

In some embodiments, sensors have been described as being used to detectthe presence or absence of an analyte (e.g., H+). It will beappreciated, however, that detecting the presence or absence of ananalyte can include detecting (or measuring) the concentration of ananalyte. For example, detecting the presence or absence of H+ in aliquid sample using any of the sensors described herein can includedetecting (or measuring) the concentration of H+.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications can be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents.

What is claimed is:
 1. An analyte sensor, comprising: a first solidstate working electrode having disposed thereon a redox-active moietyexhibiting an oxidation potential and/or a reduction potential that issensitive to the presence of an analyte, said first solid state workingelectrode doped p-type; and a second solid state working electrodehaving disposed thereon a redox-active moiety exhibiting an oxidationpotential and/or a reduction potential that is insensitive to thepresence of said analyte, said second solid state working electrodedoped n-type or p-type.
 2. The analyte sensor of claim 1, wherein saidfirst solid state working electrode has a resistivity that is greaterthan or equal to about 1 Ω-cm.
 3. The analyte sensor of claim 1, whereinsaid second solid state working electrode has a resistivity that isgreater than or equal to about 5 μΩ-cm.
 4. The analyte sensor of claim1, wherein said first and second solid state working electrodes areformed of a semiconductor.
 5. The analyte sensor of claim 4, whereinsaid semiconductor is silicon.
 6. The analyte sensor of claim 4, whereinsaid second solid state working electrode is doped n-type and has aresistivity that is greater than or equal to about 1 Ω-cm.
 7. Theanalyte sensor of claim 6, wherein the resistivity of the second solidstate working electrode is between about 1 Ω-cm and 90 Ω-cm.